Crystalline salt forms of boc-d-arg-dmt-lys-(boc)-phe-nh2

ABSTRACT

Disclosed are various crystalline salt forms of Boc-D-Arg-DMT-Lys(Boc)-Phe-NH2.

RELATED APPLICATIONS

This application is the U.S. National Stage of International PatentApplication No. PCT/US2018/025990, filed Apr. 4, 2018, which claims thebenefit of priority to U.S. Provisional Patent Application Ser. No.62/481,766, filed Apr. 5, 2017.

BACKGROUND

Through oxidative phosphorylation, mitochondria convert nutrients andoxygen into adenosine triphosphate (ATP), the chemical transporter ofenergy in most aerobic organisms. The electron transport chain (ETC) ofthe mitochondria represent the primary source of ATP, as well as asource of reactive oxygen species (ROS). Mitochondrial dysfunction in acell results in less ATP production and, as a result, insufficientenergy to maintain the cell. Such dysfunction also results in excessiveROS production, spiraling cellular injury, and ultimately apoptosis ofthe cell. Accordingly, mitochondrial dysfunction is a key elementbelieved to be at the root of a variety of serious, debilitatingdiseases.

Natural antioxidants, such as coenzyme Q and vitamin E, have been shownto provide some protection of the cell from damage induced by theelevated ROS levels associated with mitochondrial dysfunction. However,antioxidants or oxygen scavengers have also been shown to reduce ROS tounhealthy levels and may not reach the ETC in sufficient concentrationsto correct the mitochondrial imbalance. Therefore, there is a need fornovel compounds that can selectively target the ETC, restore efficientoxidative phosphorylation, and thereby address mitochondrial disease anddysfunction.

SUMMARY

Disclosed are various crystalline salt forms ofBoc-D-Arg-DMT-Lys(Boc)-Phe-NH₂.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a XRPD pattern of a hydrochloride salt of Compound I.

FIG. 2 depicts a XRPD pattern of a tosylate salt of Compound I.

FIG. 3 depicts a XRPD pattern of a mesylate salt of Compound I.

FIG. 4 depicts a XRPD pattern of an oxalate salt of Compound I.

FIG. 5 depicts a XRPD pattern of a L-tartaric acid salt of Compound I.

FIG. 6 depicts a XRPD pattern of a fumarate salt of Compound I.

FIG. 7 depicts a XRPD pattern of a benzoic acid salt of Compound I.

FIG. 8 depicts a XRPD pattern of a succinate salt of Compound I.

FIG. 9 depicts a XRPD pattern of a hydrochloride salt of Compound Icrystallized from Methanol:2-Propanol (75%:25% v/v).

FIG. 10 depicts a XRPD pattern of a hydrochloride salt of Compound Icrystallized from Methanol.

FIG. 11 depicts a XRPD pattern of a tosylate salt of Compound Icrystallized from Acetone.

FIG. 12 depicts a XRPD pattern of a mesylate salt of Compound Icrystallized from Acetone.

FIG. 13 depicts a XRPD pattern of an oxalate salt of Compound Icrystallized from Acetone.

FIG. 14 depicts a XRPD pattern of a benzoate salt of Compound Icrystallized from Methanol.

DETAILED DESCRIPTION

The present invention features salts of Compound I:

(I; Boc-D-Arg-DMT-Lys(Boc)-Phe-NH₂), wherein Boc-representstert-butyl-O—C(O)—.

Compound I is a synthetic precursor of Compound II:

(II; MTP-131; D-Arg-DMT-Lys-Phe-NH₂) or a salt thereof. Compound II hasbeen shown to affect the mitochondrial disease process by helping toprotect organs from oxidative damage caused by excess ROS production,and to restore normal ATP production.

A crystalline form of a salt of Compound I can be used tomodulate/improve the physicochemical properties of the compound,including but not limited to solid state properties (e.g.,crystallinity, hygroscopicity, melting point, or hydration),pharmaceutical properties (e.g., solubility/dissolution rate, stability,or compatibility), as well as crystallization characteristics (e.g.,purity, yield, or morphology).

In certain embodiments, the polymorph of the crystalline salt ischaracterized by X-ray powder diffraction (XRPD). θ represents thediffraction angle, measured in degrees. In certain embodiments, thediffractometer used in XRPD measures the diffraction angle as two timesthe diffraction angle θ. Thus, in certain embodiments, the diffractionpatterns described herein refer to X-ray intensity measured againstangle 2θ.

In certain embodiments, a crystalline salt of Compound (I) is notsolvated (e.g., the crystal lattice does not comprise molecules of asolvent). In certain alternative embodiments, a crystalline salt ofCompound (I) is solvated. In some cases, the solvent is water.

In one aspect, the invention features a crystalline form of Compound Iwhich has characteristic peaks in the X-ray powder diffraction (XRPD)pattern as shown in any one of FIGS. 9-14.

In another aspect, the invention features a crystalline form of CompoundI which has characteristic peaks in the X-ray powder diffraction (XRPD)pattern at values of two theta (° 2θ) as shown in any one of Tables A-F.

The relative intensity, as well as the two theta value, of each peak inTables A-F, as well as FIGS. 9-14, may change or shift under certainconditions, although the crystalline form is the same. One of ordinaryskill in the art should be able readily to determine whether a givencrystalline form is the same crystalline form as described in one ofTables A-F as well as FIGS. 9-14 by comparing their XRPD data.

In another aspect, the invention features a crystalline form of ahydrochloride salt of Compound I, which has characteristic peaks in theX-ray powder diffraction (XRPD) pattern as shown in FIG. 9.

In yet another aspect, the invention features a crystalline form of ahydrochloride salt salt of Compound I, which has characteristic peaks inthe X-ray powder diffraction (XRPD) pattern as shown in Table A.

In another aspect, the invention features a crystalline form of ahydrochloride salt of Compound I, which has characteristic peaks in theX-ray powder diffraction (XRPD) pattern at values of two theta (° 2θ) of3.8, 4.3, 9.8, 14.6, 18.0, 18.8, 20.9, and 22.7.

In another aspect, the invention features a crystalline form of ahydrochloride salt of Compound I, which has characteristic peaks in theX-ray powder diffraction (XRPD) pattern at values of two theta (° 2θ) of3.8, 4.3, 6.5, 7.3, 9.8, 13.3, 14.2, 14.6, 16.1, 16.9, 18.0, 18.8, 19.1,19.7, 20.1, 20.5, 20.9, 22.0, 22.7, 23.2, 24.0, 25.2, and 25.9.

In another aspect, the invention features a crystalline form of ahydrochloride salt of Compound I, which has characteristic peaks in theX-ray powder diffraction (XRPD) pattern as shown in FIG. 10.

In yet another aspect, the invention features a crystalline form of ahydrochloride salt salt of Compound I, which has characteristic peaks inthe X-ray powder diffraction (XRPD) pattern as shown in Table B.

In another aspect, the invention features a crystalline form of ahydrochloride salt of Compound I, which has characteristic peaks in theX-ray powder diffraction (XRPD) pattern at values of two theta (° 2θ) of3.7, 4.4, 6.6, 9.7, 14.8, 18.0, 18.5, 18.8, 19.1, 20.9, and 22.7.

In another aspect, the invention features a crystalline form of ahydrochloride salt of Compound I, which has characteristic peaks in theX-ray powder diffraction (XRPD) pattern at values of two theta (° 2θ) of3.7, 4.4, 6.6, 7.4, 9.7, 10.6, 13.2, 14.1, 14.8, 16.7, 18.0, 18.5, 18.8,19.1, 19.5, 19.8, 20.1, 20.6, 20.9, 21.3, 22.0, 22.7, 23.1, and 24.0.

In yet another aspect, the invention features a crystalline form of atosylate salt of Compound I, which has characteristic peaks in the X-raypowder diffraction (XRPD) pattern as shown in FIG. 11.

In yet another aspect, the invention features a crystalline form of atosylate salt of Compound I, which has characteristic peaks in the X-raypowder diffraction (XRPD) pattern as shown in Table C.

In another aspect, the invention features a crystalline form of atosylate salt of Compound I, which has characteristic peaks in the X-raypowder diffraction (XRPD) pattern at values of two theta (° 2θ) of 5.2,8.9, 14.4, 17.3, 18.8, 19.5, and 21.0.

In another aspect, the invention features a crystalline form of atosylate salt of Compound I, which has characteristic peaks in the X-raypowder diffraction (XRPD) pattern at values of two theta (° 2θ) of 5.2,8.9, 10.8, 13.4, 14.4, 16.0, 17.3, 18.8, 19.5, 21.0, 23.3, and 24.6.

In yet another aspect, the invention features a crystalline form of amesylate salt of Compound I, which has characteristic peaks in the X-raypowder diffraction (XRPD) pattern as shown in FIG. 12.

In yet another aspect, the invention features a crystalline form of amesylate salt of Compound I, which has characteristic peaks in the X-raypowder diffraction (XRPD) pattern as shown in Table D.

In another aspect, the invention features a crystalline form of amesylate salt of Compound I, which has characteristic peaks in the X-raypowder diffraction (XRPD) pattern at values of two theta (° 2θ) of 5.4,13.4, 14.8, 15.8, 17.6, 19.0, and 21.3.

In another aspect, the invention features a crystalline form of amesylate salt of Compound I, which has characteristic peaks in the X-raypowder diffraction (XRPD) pattern at values of two theta (° 2θ) of 5.4,10.8, 13.4, 14.8, 15.8, 17.6, 19.0, 19.7, 21.3, 22.3. 24.1, and 25.7.

In yet another aspect, the invention features a crystalline form of anoxalate salt of Compound I, which has characteristic peaks in the X-raypowder diffraction (XRPD) pattern as shown in FIG. 13.

In yet another aspect, the invention features a crystalline form of anoxalate salt of Compound I, which has characteristic peaks in the X-raypowder diffraction (XRPD) pattern as shown in Table E.

In another aspect, the invention features a crystalline form of anoxalate salt of Compound I, which has characteristic peaks in the X-raypowder diffraction (XRPD) pattern at values of two theta (° 2θ) of 7.8,10.1, 12.8, 17.8, 18.5, 19.9, and 22.3.

In another aspect, the invention features a crystalline form of anoxalate salt of Compound I, which has characteristic peaks in the X-raypowder diffraction (XRPD) pattern at values of two theta (° 2θ) of 4.1,7.2, 7.8, 8.1, 10.1, 12.0, 12.8, 13.3, 14.5, 14.9, 17.8, 18.1. 18.5,19.9, 20.4, 21.9, 22.0, 22.3, and 23.5.

In yet another aspect, the invention features a crystalline form of abenzoate salt of Compound I, which has characteristic peaks in the X-raypowder diffraction (XRPD) pattern as shown in FIG. 14.

In yet another aspect, the invention features a crystalline form of abenzoate salt of Compound I, which has characteristic peaks in the X-raypowder diffraction (XRPD) pattern as shown in Table F.

In another aspect, the invention features a crystalline form of abenzoate of Compound I, which has characteristic peaks in the X-raypowder diffraction (XRPD) pattern at values of two theta (° 2θ) of 3.7,4.4, 14.1, 18.1, 18.9, 20.7, 22.3, and 24.3.

In another aspect, the invention features a crystalline form of abenzoate salt of Compound I, which has characteristic peaks in the X-raypowder diffraction (XRPD) pattern at values of two theta (° 2θ) of 3.7,4.4, 6.7, 9.9, 13.3, 13.7, 14.1, 15.8, 17.2, 18.1, 18.4, 18.9, 19.5,20.7, 20.9, 21.6, 22.3, and 24.3.

The term “substantially pure” as used herein, refers to a crystallinepolymorph that is greater than 90% pure, meaning that contains less than10% of any other compound, including the corresponding amorphouscompound or an alternative polymorph of the crystalline salt.Preferably, the crystalline polymorph is greater than 95% pure, or evengreater than 98% pure.

In one embodiment, the present invention features a crystalline form ofCompound I which has characteristic peaks in the X-ray powderdiffraction (XRPD) pattern as shown in any one of FIGS. 1-8 and which issubstantially pure. For example, the crystalline form can be at least90% pure, preferably at least 95% pure, or more preferably at least 98%pure.

In another embodiment, the present invention features a crystalline formof Compound I which has characteristic peaks in the X-ray powderdiffraction (XRPD) pattern at values of two theta (° 2θ) as shown in anyone of Tables 1-8 and which is substantially pure. For example, thecrystalline form can be at least 90% pure, preferably at least 95% pure,or more preferably at least 98% pure.

In another aspect, the invention relates to preparing compound (II) or asalt thereof (e.g., the tri-HCl salt) from compound (I). In someembodiments, the compound (II) is obtained via deprotection of acrystalline form of compound (I). In some embodiments, the deprotectioncomprises preparing a mixture (e.g., a slurry) of a crystalline form ofcompound (I) and a scavenger in a solvent. In some embodiments, thescavenger is triisopropylsilane. In some embodiments the solvent is2,2,2-trifluoroethanol. In some embodiments, the deprotection furthercomprises addition of an acid. In some embodiments, the acid isconcentrated hydrochloric acid (e.g., 5-6 M HCl).

Methods of Making the Crystalline Salts

In certain embodiments, the invention relates to a method for thepreparation of a crystalline salt of compound (I), comprising a)providing a freebase mixture of compound (I) in a first organic solvent;b) contacting the freebase mixture with a reagent solution comprising anacid and optionally a second organic solvent under conditions sufficientto form a mixture comprising a salt of compound (I); and c)crystallizing the salt of compound (I) from the mixture comprising thesalt of compound (I).

In certain embodiments, the invention relates to a method for thepreparation of a crystalline salt of compound (I), comprising a)providing a first salt mixture of compound (I) in a first organicsolvent; b) contacting the first salt mixture with a reagent solutioncomprising an acid and optionally a second organic solvent underconditions sufficient to form a mixture comprising a second salt ofcompound (I); and c) crystallizing the second salt of compound (I) fromthe mixture comprising the second salt of compound (I).

In certain embodiments, the invention relates to a method for thepreparation of a crystalline salt of a compound having the structure offormula (II), comprising a) providing a first mixture comprising aprotected form of compound (I) in a first organic solvent; b) contactingthe first mixture with a reagent solution comprising an acid andoptionally a second organic solvent under conditions sufficient todeprotect the protected form of compound (I) and to form a mixturecomprising a salt of compound (II); and c) crystallizing the salt ofcompound (II) from the mixture comprising the salt of compound (II). Incertain embodiments, the mixture comprising a salt of compound (I)formed in step b) is a solution. In certain embodiments, the mixtureformed in step b) is a slurry or a suspension.

In certain embodiments, the mixture comprising the salt of compound (I)or (II) is a solution, and the step of crystallizing the salt from themixture comprises bringing the solution to supersaturation to cause thesalt of compound (I) or (II) to precipitate out of solution.

In certain embodiments, bringing the mixture comprising the salt ofcompound (I) or (II) to supersaturation comprises the slow addition ofan anti-solvent, such as heptanes, hexanes, ethanol, or another polar ornon-polar liquid miscible with the organic solvent, allowing thesolution to cool (with or without seeding the solution), reducing thevolume of the solution, or any combination thereof. In certainembodiments, bringing the mixture comprising the salt of compound (I) or(II) to supersaturation comprises adding an anti-solvent, cooling thesolution to ambient temperature or lower, and reducing the volume of thesolution, e.g., by evaporating solvent from the solution. In certainembodiments, allowing the solution to cool may be passive (e.g.,allowing the solution to stand at ambient temperature) or active (e.g.,cooling the solution in an ice bath or freezer).

In certain embodiments, the preparation method further comprisesisolating the salt crystals, e.g. by filtering the crystals, bydecanting fluid from the crystals, or by any other suitable separationtechnique. In further embodiments, the preparation method furthercomprises washing the crystals.

In certain embodiments, the preparation method further comprisesinducing crystallization. The method can also comprise the step ofdrying the crystals, for example under reduced pressure. In certainembodiments, inducing precipitation or crystallization comprisessecondary nucleation, wherein nucleation occurs in the presence of seedcrystals or interactions with the environment (crystallizer walls,stirring impellers, sonication, etc.).

In certain embodiments, the freebase mixture of compound (I) in a firstorganic solvent is a slurry. In certain embodiments, the freebasemixture of compound (I) in a first organic solvent is a solution.

In certain embodiments, the first organic solvent and the second organicsolvent, if present, comprise acetone, anisole, methanol, 1-butanol,2-butanone, iso-butanol, tert-butanol, sec-butanol, cyclopentyl methylether (CPME), benezotrifluoride (BTF), 1-propanol, 2-propanol (IPA),water, dichloromethane, anisole, acetonitrile, ethylene glycol,tert-butyl methyl ether (t-BME), DMSO, ethylene glycol, toluene,tetrahydrofuran (THF), heptane, acetonitrile, N,N-dimethylacetamide(DMA), dimethylformamide (DMF), dimethylsulfoxide (DMSO), 1,4-dioxane,2-ethoxy ethanol, heptane, isopropyl acetate, methyl acetate, 2-methylTHF, methyl isobutyl ketone (MIBK), 1-propanol, ethanol, ethyl acetate,hexanes, methyl acetate, isopropyl acetate, methylethyl ketone,1,4-dioxane, methyl cyclohexane, N-methyl-2-pyrrolidone (NMP), or anycombination thereof.

In certain embodiments, the first organic solvent and the second organicsolvent, if present, are the same. In alternative embodiments, the firstorganic solvent and the second organic solvent, if present, aredifferent.

In certain embodiments, washing the crystals comprises washing with aliquid selected from anti-solvent, acetonitrile, ethanol, heptanes,hexanes, methanol, tetrahydrofuran, toluene, water, or a combinationthereof. As used herein, “anti-solvent” means a solvent in which thesalt crystals are insoluble, minimally soluble, or partially soluble. Inpractice, the addition of an anti-solvent to a solution in which thesalt crystals are dissolved reduces the solubility of the salt crystalsin solution, thereby stimulating precipitation of the salt. In certainembodiments, the crystals are washed with a combination of anti-solventand the organic solvent. In certain embodiments, the anti-solvent iswater, while in other embodiments it is an alkane solvent, such ashexane or pentane, or an aromatic hydrocarbon solvent, such as benzene,toluene, or xylene. In certain embodiments, the anti-solvent is ethanol.

In certain embodiments, washing the crystals comprises washingcrystalline compound (I) with a solvent or a mixture of one or moresolvents, which are described above. In certain embodiments, the solventor mixture of solvents is cooled prior to washing.

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

EXAMPLES Materials and Methods X-Ray Powder Diffraction (XRPD)

Powder x-ray diffraction experiments were performed on a PANalyticalX'Pert Pro X-ray Diffractometer, scanning the samples between 3 and 35°2θ. Material was loaded into a 96-well plate with mylar film as thebase. The samples were then loaded into the plate holder of aPANalytical X'Pert Pro X-ray Diffractometer running in transmission modeand analyzed, using the following experimental conditions:

Raw Data Origin: XRPD measurement (*.XRPDML) Scan Axis: Gonio StartPosition [°2θ]: 3.0066 End Position [°2θ]: 34.9866 Step Size [°2θ]:0.0130 Scan Step Time [s]: 18.8700 Scan Type: Continuous PSD Mode:Scanning PSD Length [°2θ]: 3.35 Offset [°2θ]: 0.0000 Divergence SlitType: Fixed Divergence Slit Size [°]: 1.0000 Specimen Length [mm]: 10.00Measurement Temperature [° C.]: 25.00 Anode Material: Cu K-Alpha1 [Å]:1.54060 K-Alpha2 [Å]: 1.54443 K-Beta [Å]: 1.39225 K-A2/K-A1 Ratio:0.50000 Generator Settings: 40 mA, 40 kV Diffractometer Type:0000000011154173 Diffractometer Number: 0 Goniometer Radius [mm]: 240.00Dist. Focus-Diverg. Slit [mm]: 91.00 Incident Beam Monochromator: NoSpinning: No

Polarized Light Microscopy (PLM)

The presence of birefringence was determined using an Olympus BX50polarizing microscope, equipped with a Motic camera and image capturesoftware (Motic Images Plus 2.0). All images were recorded using the 20×objective, unless otherwise stated.

Thermogravimetric/Differential Thermal Analysis (TG/DTA)

Approximately 5 mg of material was weighed into an open aluminium panand loaded into a simultaneous thermogravimetric/differential thermalanalyzer (TG/DTA) and held at room temperature. The sample was thenheated at a rate of 10° C./min from 20° C. to 300° C. during which timethe change in sample weight was recorded along with any differentialthermal events (DTA). Nitrogen was used as the purge gas, at a flow rateof 300 cm³/min.

Differential Scanning Calorimetry (DSC)

Approximately 5 mg of material was weighed into an aluminium DSC pan andsealed non-hermetically with a pierced aluminium lid. The sample pan wasthen loaded into a Seiko DSC6200 (equipped with a cooler) and held at20° C. Once a stable heat-flow response was obtained, the sample andreference were heated to ca. 180° C. at a scan rate of 10° C./min andthe resulting heat flow response monitored. Nitrogen was used as thepurge gas, at a flow rate of 50 cm3/min.

Karl Fischer Coulometric Titration (KF)

Approximately 10 mg of solid material was accurately weighed into avial. The solid was then dissolved in ca. 1 mL or 5 mL of pre-titratedHydranal solution, sonicating for ca. 5-10 min. The solution wasmanually introduced into the titration cell of a Mettler Toledo C30Compact Titrator and the weight of the solid entered on the instrument.

1H Nuclear Magnetic Resonance Spectroscopy (1H NMR)

1H-NMR spectroscopic experiments were performed on a Bruker AV500(frequency: 500 MHz). Experiments were performed in d6-dimethylsulfoxideand each sample was prepared to ca. 10 mM concentration.

Gravimetric Vapour Sorption (GVS)

Approximately 15 mg of sample was placed into a mesh vapour sorptionbalance pan and loaded into an IGASorp Moisture Sorption Analyserbalance by Hiden Analytical. The sample was subjected to a rampingprofile from 40-90% relative humidity (RH) at 10% increments,maintaining the sample at each step until a stable weight had beenachieved (98% step completion). After completion of the sorption cycle,the sample was dried using the same procedure to 0% RH, then subjectedto a second ramping profile from 0-90% relative humidity. Aftercompletion of the second sorption cycle, the sample was dried using thesame procedure to 0% RH, and finally taken back to the starting point of40% RH. The weight change during the sorption/desorption cycles wereplotted, allowing for the hygroscopic nature of the sample to bedetermined.

High Performance Liquid Chromatography-Ultraviolet Detection (HPLC-UV)

Column: Aeris Peptide C18 3.6 μm 250×4.6 mm column

Mobile Phase A: 0.05% TFA in deionized water

Mobile Phase B: 0.05% TFA in acetonitrile

Diluent: Water:Acetonitrile (90:10 v/v)

Flow Rate: 1.0 mL/min

-   -   Runtime: 32 minutes    -   Column Temperature: 30° C.    -   Autosampler Temperature: 5° C.    -   Injection Volume: 30 μL    -   Detection: 220 nm    -   Sample Concentration: 0.5 mg/mL

Gradient program:

Time/min Solvent B (%) 0.00 5 15.00 25 20.00 50 25.00 90 27.00 90 27.105 32.00 5

Example 1. Experimental Approximate Solubility Assessment ofBoc-D-Arg-DMT-Lys(Boc)-Phe-NH₂

The solubility screen was carried out as follows:

-   -   Approximately 20 mg of Boc-D-Arg-DMT-Lys(Boc)-Phe-NH₂ was        weighed out into each vial.    -   Each solvent/solvent mixture was added to the appropriate vial        in 5 volume aliquots (100 μL).    -   In between additions, the sample was stirred at 50° C. (35° C.        for DCM).    -   If 2000 μL of solvent was added without dissolution of the        material, solubility was calculated to be below this point.    -   XRPD analysis of residual solids was carried out where        solubility was <17 mg/mL.

TABLE 1 Solvent Systems Selected for Solubility Screen Solvent ICH ClassAcetone 3 Ethanol 3 Methanol 2 2-Propanol 3 2-Butanol 3 Methyl ethylketone 3 Dichloromethane* 2 Toluene 3 Acetone:water (50:50% v/v) 3Ethanol:water (50:50% v/v) 3 Methanol:water (50:50% v/v) 22-propanol:water (50:50% v/v) 3 Tetrahydrofuran 2 Ethyl acetate 3Acetonitrile 2 Trifluoroethanol Not classified Acetonitrile:water(50:50% v/v) 2 DMSO:acetone (50:50% v/v) 3 DMSO:water (50:50% v/v) 3

Small-Scale Crystallization Trials

Cooling/Temperature Cycling Crystallizations

General Procedure:

-   -   ca. 40 mg of Boc-D-Arg-DMT-Lys(Boc)-Phe-NH₂ was weighed out into        a 2 mL vial.    -   Respective solvent was added to the appropriate vial at ambient        (ca. 22° C.).    -   The experiments were heated to 50° C. and further stirred at 50°        C.    -   The experiments were stirred at 50° C. for ca. 1 hour.    -   The experiments were cooled down to 5° C. at 0.1° C./minutes.    -   The experiments were temperature cycled between 5° C. and 40° C.        for about 16 hours of cycling.    -   For the experiments where a slurry was observed, the solid was        isolated by centrifugation using a 0.22 μm Nylon polypropylene        centrifuge filter at 5° C. and the isolated material was        analyzed by XRPD.    -   The isolated material was dried under vacuum at ca. 30-40° C.        for ca. 18 hours.

TABLE 2 Experimental Details for Small-Scale Cooling/Temperature CyclingCrystallization Trials Input Vol. of Sample Material Solvent/solventsolvent/solvent Concentration ID (mg) system system used (mL) (mg/mL) 140.07 Acetone:water 0.60 66.8 (50:50% v/v) 2 40.64 Ethanol 0.40 101.6 341.51 Methanol 0.28 150.4 4 40.13 Methanol 0.40 100.3 5 41.23Trifluoroethanol 0.10 412.3 6 40.40 Acetonitrile:water 0.27 149.6(50:50% v/v) 7 40.12 DMSO:water 0.20 200.6 (80:20% v/v)

Anti-Solvent Addition/Cooling/Temperature Cycling Crystallizations

General Procedure:

-   -   ca. 40 mg of Boc-D-Arg-DMT-Lys(Boc)-Phe-NH₂ was weighed out into        a 2 mL vial.    -   Respective solvent was added to the appropriate vial at ambient        (ca. 22° C.).    -   The experiments were heated to 50° C. and further stirred at 50°        C.    -   A clear solution was observed at 50° C. for each experiment.    -   The respective anti-solvent was added to the appropriate        experiment at 50° C.    -   The experiments were stirred at 50° C. for ca. 1 hour.    -   The experiments were cooled down to 5° C. at 0.1° C./minutes.        The experiments were temperature cycled between 5° C. and 40° C.        for about 16 hours of cycling.    -   In the experiments where a thick slurry/thick precipitation was        observed, the respective solvent/anti-solvent (same ratio) was        added to improve mixing at 5° C.    -   For the experiments where a slurry was observed, the solid was        isolated by centrifugation using a 0.22 μm Nylon polypropylene        centrifuge filter at 5° C. and the isolated material was        analyzed by XRPD.

TABLE 3 Experimental Details for Anti-solvent additionCooling/Temperature Cycling Crystallizations Vol. of Vol. of solventAnti- Input Solvent/ system solvent Total Concen- % Sample Solid solventAnti- used used Volume tration % Anti- ID (mg) system solvent (mL) (mL)(mL) (mg/mL) Solvent Solvent 1 41.63 Methanol Acetonitrile 0.28 0.1 0.38110.7 73.4 26.6 2 41.08 Methanol EA 0.28 0.1 0.38 108.1 73.7 26.3 340.92 Methanol THF 0.28 0.1 0.38 107.7 73.7 26.3 4 40.5 Methanol Acetone0.28 0.1 0.38 106.6 73.7 26.3 5 40.15 Methanol MEK 0.28 0.1 0.38 105.773.7 26.3 6 39.99 Methanol Toluene 0.28 0.1 0.38 105.2 73.7 26.3 7 40.16Methanol Heptane 0.28 0.1 0.38 105.7 73.7 26.3 8 41.2 Ethanol EA 0.290.1 0.39 105.6 74.4 25.6 9 41.075 Ethanol THF 0.29 0.1 0.39 105.3 74.425.6 10 42.93 Ethanol Acetone 0.29 0.1 0.39 110.1 74.4 25.6 11 41.005Ethanol MEK 0.29 0.1 0.39 105.1 74.4 25.6 12 40.78 Ethanol Toluene 0.290.1 0.39 104.6 74.4 25.6 13 42.045 Ethanol Heptane 0.29 0.1 0.39 107.874.4 25.6 14 39.295 Ethanol TBME 0.29 0.1 0.39 100.8 74.4 25.6 15 41.6Ethanol Acetonitrile 0.29 0.1 0.39 106.7 74.4 25.6 16 40.16Trifluoroethanol EA 0.1 0.1 0.2 200.8 50.0 50.0 17 40.095Trifluoroethanol THF 0.1 0.1 0.2 200.5 50.0 50.0 18 42.545Trifluoroethanol Acetone 0.1 0.1 0.2 212.7 50.0 50.0 19 41.175Trifluoroethanol MEK 0.1 0.1 0.2 205.9 50.0 50.0 20 41.365Trifluoroethanol Toluene 0.1 0.1 0.2 206.8 50.0 50.0 21 39Trifluoroethanol Heptane 0.1 0.1 0.2 195.0 50.0 50.0 22 40.2Trifluoroethanol TBME 0.1 0.1 0.2 201.0 50.0 50.0 23 41.48Trifluoroethanol Acetonitrile 0.1 0.1 0.2 207.4 50.0 50.0

Anti-Solvent Addition Crystallizations

General Procedure:

-   -   ca. 40 mg of Boc-D-Arg-DMT-Lys(Boc)-Phe-NH₂ was weighed out into        a 2 mL vial.    -   Respective solvent was added to the appropriate vial at ambient        (ca. 22° C.).    -   The experiments were heated to 50° C. and stirred at 50° C.    -   Clear solution was observed at 50° C. for each experiment.    -   For addition of anti-solvent at 5° C.:        -   The experiments were cooled down to 5° C. at 0.1° C./minutes            and temperature cycled between 5° C. to 40° C. at 0.1°            C./minutes for ca. 18 hours cycle.        -   At 5° C., respective anti-solvent was added to the            appropriate experiment.        -   Further the experiments were stirred at 5° C. for ca 2            hours.    -   For anti-solvent addition at 50° C.:        -   To the clear solutions at 50° C., respective anti-solvents            were added.        -   The experiments were stirred at 50° C. for ca. 2 hours.        -   The experiments were cooled down to 5° C. at 0.1°            C./minutes. The experiments were temperature cycled between            5° C. and 40° C. for about 18 hour cycles.    -   In the experiments where a thick slurry/thick precipitation was        observed, the respective solvent/anti-solvent (same ratio) was        added to ensure a stirrable slurry at 5° C.    -   For the experiments where a flowable slurry was observed, the        solid was isolated by centrifugation using 0.22 μm Nylon        polypropylene centrifuge tube at 5° C. and the isolated material        was analyzed by XRPD.    -   The isolated material was dried under vacuum at 40° C. for ca.        24 hours.

TABLE 4 Experiment Details for Anti-Solvent Addition CrystallizationsVol. of Vol. of solvent Anti- Input Solvent/ system solvent TotalConcen- % Sample Material solvent Anti- used used Volume tration % Anti-ID (mg) system solvent Temperature (mL) (mL) (mL) (mg/mL) SolventSolvent 1 39.83 Methanol Acetonitrile 50° C. 0.27 0.81 1.08 36.9 25 75 239.61 EA 0.27 0.81 1.08 36.7 25 75 3 39.48 THE 0.27 0.81 1.08 36.6 25 754 40.49 Acetone 0.27 0.81 1.08 37.5 25 75 5 39.8 MEK 0.27 0.81 1.08 36.925 75 6 40.35 Toluene 0.27 0.81 1.08 37.4 25 75 7 40.53 TBME 0.27 0.811.08 37.5 25 75 8 39.51 Ethanol Acetonitrile 50° C. 0.29 0.87 1.16 34.125 75 9 41.04 EA 0.29 0.87 1.16 35.4 25 75 10 41.18 THE 0.29 0.87 1.1635.5 25 75 11 42.05 Acetone 0.29 0.87 1.16 36.3 25 75 12 40.1 MEK 0.290.87 1.16 34.6 25 75 13 41.31 Toluene 0.29 0.87 1.16 35.6 25 75 14 41.27TBME 0.29 0.87 1.16 35.6 25 75 15 39.7 Heptane 0.29 0.87 1.16 34.2 25 7516 40.69 Methanol Acetonitrile  5° C. 0.27 0.81 1.08 37.7 25 75 17 40.82EA 0.27 0.81 1.08 37.8 25 75 18 41.28 THE 0.27 0.81 1.08 38.2 25 75 1940.29 Acetone 0.27 0.81 1.08 37.3 25 75 20 39 35 MEK 0.27 0.81 1.08 36.425 75 21 40.74 Toluene 0.27 0.81 1.08 37.7 25 75 22 41.68 TBME 0.27 0.811.08 38.6 25 75 23 40.96 Ethanol Acetonitrile  5° C. 0.29 0.87 1.16 35.325 75 24 40.33 EA 0.29 0.87 1.16 34.8 25 75 25 41.12 THE 0.29 0.87 1.1635.4 25 75 26 39.97 Acetone 0.29 0.87 1.16 34.5 25 75 27 40.31 MEK 0.290.87 1.16 34.7 25 75 28 40.85 Toluene 0.29 0.87 1.16 35.2 25 75 29 40.72TBME 0.29 0.87 1.16 35.1 25 75 30 39.7 Heptane 0.29 0.87 1.16 34.2 25 75

Seeded Cooling Crystallizations using Solvent/Anti-Solvent Mixtures

General Procedure:

-   -   ca. 40 mg of Boc-D-Arg-DMT-Lys(Boc)-Phe-NH₂ was weighed out into        a 2 mL vial.    -   Respective solvent was added to the appropriate vial at ambient.    -   The experiments were heated to 50° C. and further stirred at 50°        C.        -   A clear solution was observed at 50° C. for each experiment.    -   The respective anti-solvent was added to the appropriate        experiment at 50° C. Clear solution was observed. The        experiments were seeded using crystalline Form 1.        -   Seed persisted in all the crystallizations and turbidity was            observed.    -   The experiments were stirred at 50° C. for ca. 1 hour.        -   Further nucleation was observed in every experiment.    -   The experiments were cooled down to 5° C. at 0.1° C./minute. The        experiments were further stirred at 5° C.    -   In the experiments where a thick slurry/thick precipitation was        observed, the respective solvent/anti-solvent (same ratio) was        added to improve mixing at 5° C.    -   The experiments were isolated by centrifugation using a 0.22 μm        Nylon polypropylene centrifuge filter at 5° C. and the isolated        material was analyzed by XRPD.    -   Dried under vacuum at 35° C. to 40° C. for ca. 24 hours.

TABLE 5 Experimental Details for Seeded Cooling Crystallizations usingSolvent/Anti-solvent Mixtures Vol. of Vol. of Anti- % anti- Inputsolvent solvent Total solvent/ Sample material Anti- used used VolumeConcentration % co- ID (mg) Solvent solvent (mL) (mL) (mL) (mg/mL)Solvent solvent 1 40.3 Methanol Acetone 0.27 0.090 0.360 111.9 75 25 240 Methanol 2-propanol 0.27 0.090 0.360 111.1 75 25 3 39.9 Methanol2-butanol 0.27 0.090 0.360 110.8 75 25 4 41.5 Methanol TBME 0.27 0.0900.360 115.3 75 25 5 42.05 Methanol Ethanol 0.20 0.200 0.400 105.1 50 506 41.5 Ethanol 2-propanol 0.29 0.097 0.387 107.3 75 25 7 39.9 Ethanol2-butanol 0.29 0.097 0.387 103.2 75 25

Scale-up Seeded Cooling Crystallizations Using Solvent/Anti-SolventMixture

In order to reproduce the most promising small-scale crystallizations,to assess repeatability and obtain further material forcharacterization, scale-up crystallizations were carried out. Thefollowing procedure was used:

-   -   ca. 250 mg of Boc-D-Arg-DMT-Lys(Boc)-Phe-NH₂ was weighed out        into a 20 mL scintillation vial.    -   Respective solvent was added to the appropriate vial at ambient.    -   The experiments were heated to 50° C. and further stirred at 50°        C.        -   A clear solution was observed at 50° C. for each experiment.    -   The respective anti-solvent was added to the appropriate        experiment at 50° C. Clear solution was observed. The        experiments were seeded using crystalline Form 1.        -   Seed persisted in all the crystallizations and turbidity was            observed.    -   The experiments were stirred at 50° C. for ca. 1 hour.    -   Further nucleation was observed in every experiment.    -   The experiments were cooled down to 5° C. at 0.1° C./minutes.        The experiments were further stirred at 5° C.    -   The experiments were isolated at 5° C. by filtering over a        Buchner funnel using Whatmann filter paper (Grade 597).    -   The isolated material from each batch was air dried for ca. 18        hours and further dried under vacuum at 35° C. to 40° C. for ca.        24 hours.

TABLE 6 Experimental Details for Scale-up Seeded CoolingCrystallizations Vol. of Vol. of Anti- Input Concentration solventsolvent Total Concentration % Sample material Anti- in solvent used usedVolume after ASA % Anti- ID (mg) Solvent solvent (mg/mL) (mL) (mL) (mL)(mg/mL) Solvent solvent 1 254 Methanol 100 2.543 2.543 100.00 100 2 250Methanol Acetonitrile 150 1.667 0.556 2.222 112.50 75 25 3 256 MethanolTHF 150 1.708 5.125 6.834 37.50 25 75 4 252 Ethanol Acetone 140 1.8045.412 7.216 35.00 25 75

Primary Salt Screening

General Procedure

-   -   ca. 25 mg of Boc-D-Arg-DMT-Lys(Boc)-Phe-NH₂ was weighed out into        a 2 mL vial.    -   Respective solvent was added to the appropriate vial at ambient.    -   The experiments were stirred at 50° C. for ca. 1 hour.        -   For experiments using acetone as a solvent, a slurry was            observed.        -   For all other solvents, initially clear solutions were            observed but during further stirring at 50° C. precipitation            were observed. A slurry was present.    -   Further respective solvent was added to the appropriate        experiments either to dissolve the precipitated material or to        improve stirring.    -   The counterions were weighed (1.0 mole equivalent). To the vials        containing counterion, the respective solvent was added at        ambient.    -   The solution/slurry of counterion in the respective solvent        system was added to the appropriate vial containing the        slurry/clear solution of Boc-D-Arg-DMT-Lys(Boc)-Phe-NH₂ in the        respective solvent system at 50° C.    -   The vial containing solution/slurry of counterion was washed        with the respective solvent (50 μL) and the washings were added        to the salt formation reaction.    -   The experiments were stirred at 50° C. for ca. 1 hour.    -   In the experiments where a thick slurry was observed, the        respective solvent was added to improve mixing.    -   The experiments were cooled down to 5° C. at 0.1° C./minutes.        The experiments were temperature cycled between 5° C. and 40° C.        for ca. 16 hours of cycling.        -   At 16 hours, observations were recorded.        -   Temperature cycling was continued for a total of ca. 40            hours.        -   For the experiments where a slurry was observed, the solid            was isolated by centrifugation using a 0.22 μm Nylon            polypropylene centrifuge filter at 5° C.            -   The isolated material was dried under vacuum at 30° C.                for 2 hours.        -   The isolated material was analyzed by XRPD.

TABLE 7 Experimental Details for Primary Salt Screening Further solventsystem added Further Solvent either to solvent system dissolve Solventsystem Mass of Mass of added Initial or to system added to InputSolvent/ counterion to input concen- make for improve Sample Materialsolvent (mg material tration slurry counterion mixing ID (mg) systemCounterion or μL) (μL) (mg/mL) (μL) (μL) (μL) 1 26.26 Acetone HCl 2.7600 43.77 N/A 75 100 2 25.5 Acetonitrile: 2.6 130 196.15 25 50 100 water(50:50% v/v) 3 26.18 Ethanol 2.6 187 140.00 50 50 N/A 4 26.53 Methanol2.7 132 200.98 75 50 N/A 5 25.38 Acetone P-toluene 5.93 400 63.45 N/A 75N/A 6 25.23 Acetonitrile: sulfonic 5.97 130 194.08 25 50 N/A water acid(50:50% v/v) 7 25.33 Ethanol 6.06 187 135.45 50 50 N/A 8 25.53 Methanol5.99 132 193.41 75 50 N/A 9 26.04 Acetone Methane 2 400 65.10 N/A 75 N/A10 25.5 Acetonitrile: sulfonic 2 130 196.15 25 50 N/A water acid (50:50%v/v) 11 26.04 Ethanol 2 187 139.25 50 50 N/A 12 26.44 Methanol 2.1 132200.30 75 50 N/A 13 25.74 Acetone Oxalic 2.95 400 64.35 N/A 75 N/A 1426.18 Acetonitrile: acid 2.99 130 201.38 25 50 N/A water (50:50% v/v) 1526.61 Ethanol 2.98 187 142.30 50 50 N/A 16 25.81 Methanol 2.86 132195.53 75 50 N/A

TABLE 8 Experimental Details for Primary Salt Screening Further solventsystem Further added solvent Solvent either to system system dissolveSolvent added Mass of Mass of added or to system to Input Solvent/counter- to input Initial make for improve Sample material solventCounter- ion (mg material concentration slurry counter- mixing ID (mg)system ion or μL) (μL) (mg/mL) (μL) ion (μL) (μL) 17 25.36 AcetoneL-Tartaric 4.69 400 63.40 N/A 75 N/A 18 26.29 Acetonitrile: acid 4.87130 202.23 25 50 100 water (50:50%)v/v 19 25.05 Ethanol 4.66 187 133.9650 50 100 20 25.4 Methanol 4.8 132 192.42 75 50 100 21 26.2 AcetoneFumaric 3.96 400 65.50 NA 75 N/A 22 25.26 Acetonitrile: Acid 3.67 130194.31 25 50 N/A water (50:50%)v/v 23 25.22 Ethanol 3.69 187 134.87 5050 N/A 24 25.34 Methanol 3.62 132 191.97 75 50 N/A 25 26 Acetone Benzoic3.88 400 65.00 N/A 75 100 28 26.49 Acetonitrile: acid 4.09 130 203.77 2550 N/A water (50:50%)v/v 27 25.27 Ethanol 3.78 187 135.13 50 50 N/A 2825.23 Methanol 3.86 132 191.14 75 50 N/A 29 25.2 Acetone Succinic 3.67400 63.00 N/A 75 100 30 25.75 Acetonitrile: acid 3.86 130 198.08 25 50N/A water (50:50%)v/v 31 25.24 Ethanol 3.61 187 134.97 50 50 100 3226.43 Methanol 3.88 132 203.23 75 50 N/A

Example 2. Results Characterization of Boc-D-Arg-DMT-Lys(Boc)-Phe-NH₂ byXRPD, PLM, TG/DTA, DSC, GVS, KF and HPLC-UV.

Characteristics:

-   -   predominantly amorphous by XRPD analysis.    -   non-birefringent by PLM analysis, with no clearly defined        morphology.    -   TG analysis showed a weight loss of ca. 2.79% from the outset up        to ca. 144° C., followed by weight loss of ca. 0.72%        corresponding to an endothermic event in the DTA at an onset of        ca. 144.3° C. (peak at ca. 155.2° C.).    -   DSC analysis showed a broad endothermic event from the outset up        to ca. 140° C., likely due to unbound solvent/water. A second        endotherm was observed at an onset of ca. 140.2° C. (peak at        155.6° C.).    -   GVS analysis of the indicated that        Boc-D-Arg-DMT-Lys(Boc)-Phe-NH₂, is highly hygroscopic, with a        mass increase of ca. 10% between 40-90% RH observed. The        post-GVS sample was also found to be predominantly amorphous by        XRPD.    -   contained ca. 3.61% water by KF analysis.    -   purity of 97.50% by HPLC analysis.

Approximate Solubility Assessment of Boc-D-Arg-DMT-Lys(Boc)-Phe-NH₂

The solubility assessment was estimated by a solvent addition technique,heating at 50° C. between aliquots (see Table 9). The followingobservations and results were obtained:

-   -   excellent solubility in methanol, trifluoroethanol,        acetonitrile:water (50:50% v/v) and DMSO:acetone (50:50% v/v)        giving solubility values of ≥200 mg/mL.    -   Solubility values of ca. 140 mg/mL in ethanol and ca. 100 mg/mL        in 2-propanol:water (50:50% v/v) and ethanol:water (50:50% v/v)        were also observed.    -   Moderate solubility (ca. 58 to 24 mg/mL) was obtained in        acetone:water (50:50 v/v), methanol:water (50:50 v/v), and        DMSO:water (50:50 v/v).    -   Poor solubility (≤17 mg/mL) was obtained in all other solvent        systems investigated, including acetone, dichloromethane,        2-butanol, 2-propanol, methyl ethyl ketone, toluene, THF, ethyl        acetate and acetonitrile.    -   XRPD analysis was carried out on residual solids from some of        the solvent systems showing poor solubility, after slurrying at        50° C. overnight, with diffractograms of residual solids being        predominantly amorphous.

TABLE 9 Approximate Solubility Screen Results Approximate SolubilitySolvent (50° C.)/mg/mL Acetone <10 Ethanol 140 Methanol 200 2-Propanol14 2-Butanol 17 Methyl ethyl ketone <10 Dichloromethane* <10 Toluene <10Acetone:water (50:50% v/v) 58 Ethanol:water (50:50% v/v) 100Methanol:water (50:50% v/v) 45 2-propanol:water (50:50% v/v) 100Tetrahydrofuran <10 Ethyl acetate <10 Acetonitrile <10Trifluoroethanol >200 Acetonitrile:water (50:50% v/v) 200 DMSO:acetone(50:50% v/v) 400 DMSO:water (50:50% v/v) 24 *35° C. for dichloromethane

Small-Scale Crystallization Trials

Cooling/Temperature Cycling Crystallizations

Small-scale temperature cycling crystallization trials usingBoc-D-Arg-DMT-Lys(Boc)-Phe-NH₂ were carried out in 7 different solventsystems, using ethanol, methanol, trifluoroethanol, acetone:water(50:50% v/v, Acetonitrile:water(50:50% v/v) and DMSO:water (80;20% v/v).The following results and observations were obtained from theseexperiments:

-   -   Clear solutions were observed in all of the experiments at 50°        C.    -   Within 1 hour of granulation at 50° C., nucleation followed by        crystallization was observed in experiments using methanol at        both concentrations of ca. 100 mg/mL and 150 mg/mL.    -   Observations and results are summarized in Table 10.    -   XRPD analysis showed crystallization of the material from        experiments using methanol as a solvent.    -   PLM analysis of the dried, crystalline solids from methanol (ca.        100 mg/mL and 150 mg/mL concentration) indicated that the        material was birefringent with no well-defined morphology.    -   TG/DT analysis of the dried material isolated from methanol (100        mg/mL) showed a weight loss of ca.1.39% from the outset up to        ca. 168° C. An endothermic event at an onset of 168.5° C. (peak        at 175.9° C.) followed by degradation of the material was        observed in the DTA.    -   HPLC analysis of the dried material isolated from acetone: water        (50:50% v/v) and ethanol indicated a purity value of 98.80% and        98.81% respectively.    -   HPLC analysis of the dried crystalline material isolated from        methanol using concentrations of ca. 150 mg/mL and ca. 100 mg/mL        indicated purity values of 98.24% and 98.62% respectively.

TABLE 10 Observations and Results from Cooling/Temperature CyclingCrystallizations Observation Observation after Sample Solvent/solvent attemperature ID system 50° C. cycling XRPD 1 Acetone:water Clear solutionSlurry Predominantly (50:50% v/v) amorphous 2 Ethanol Clear solutionGel-like Predominantly amorphous 3 Methanol Clear solution Very thickCrystalline slurry 4 Methanol Clear solution Slurry Crystalline Clear 5Trifluoroethanol Clear solution Clear solution solution 6Acetonitrile:water Clear solution Gel-like thin (50:50% v/v) slurry 7DMSO:water Clear solution Clear (80:20% v/v) solution

Anti-Solvent Addition/Cooling/Temperature Cycling Crystallizations

Small-scale cooling followed temperature cycling produced crystallinematerial using methanol as a solvent. In order to investigate furthersolvent systems for crystallization of Boc-D-Arg-DMT-Lys(Boc)-Phe-NH₂,cooling/temperature cycling crystallizations were carried out usingmethanol, ethanol and trifluoroethanol as the solvents and usingacetonitrile, ethyl acetate, THF, acetone, MEK, toluene and heptane asthe anti-solvents. The following results and observations were obtainedfrom these experiments:

-   -   After temperature cycling, a slurry was observed for experiments        using methanol/anti-solvent mixtures.    -   Thick precipitation was observed after temperature cycling for        most of the experiments using ethanol/anti-solvent and        trifluoroethanol/anti-solvent mixtures.    -   The wet material isolated from methanol/anti-solvent mixtures        was free flowing but the wet material from ethanol/anti-solvents        and trifluoroethanol/anti-solvents was gel-like.    -   Observations and results are summarized in Table 11 and Table 12        respectively.    -   XRPD analysis on the isolated material indicated that        crystalline material was produced from methanol/anti-solvent        mixtures and poorly crystalline to partially crystalline        material from ethanol/anti-solvent mixtures and        trifluoroethanol/anti-solvent mixtures.

TABLE 11 Observations from Anti-solvent Addition Cooling/TemperatureCycling Crystallizations Observation Observation at 50° C., after %after temperature Observation Sample Anti- % Anti- addition of cycling(at before ID Solvent solvent Solvent solvent anti-solvent 5° C.)isolation 1 Methanol Acetonitrile 73.7 26.3 Clear Slurry Slurry solution2 Methanol EA 73.7 26.3 Clear Slurry Slurry solution 3 Methanol THF 73.726.3 Clear Slurry Slurry solution 4 Methanol Acetone 73.7 26.3 ClearSlurry Slurry solution 5 Methanol MEK 73.7 26.3 Clear Slurry Slurrysolution 6 Methanol Toluene 73.7 26.3 Clear Thin Slurry Thin Slurrysolution 7 Methanol Heptane 73.7 26.3 Clear Slurry Slurry solution 8Ethanol EA 74.4 25.6 Precipitation Thick Slurry precipitation 9 EthanolTHF 74.4 25.6 Clear/slight Thick Slurry turbid precipitation 10 EthanolAcetone 74.4 25.6 Clear Thick slurry Thin slurry solution 11 Ethanol MEK74.4 25.6 Slight turbid Thick Slurry precipitation 12 Ethanol Toluene74.4 25.6 Clear Thick Slurry solution precipitation 13 Ethanol Heptane74.4 25.6 Turbid Thick Slurry precipitation 14 Ethanol TBME 74.4 25.6Thin slurry Thick Slurry precipitation 15 Ethanol Acetonitrile 74.4 25.6Clear Slurry Thin slurry solution 16 Trifluoroethanol EA 50.0 50.0 ThickThick Slurry precipitation precipitation 17 Trifluoroethanol THF 50.050.0 Thick Thick Slurry precipitation precipitation 18 TrifluoroethanolAcetone 50.0 50.0 Thick Thick Slurry precipitation precipitation 19Trifluoroethanol MEK 50.0 50.0 Slurry Thick Slurry precipitation 20Trifluoroethanol Toluene 50.0 50.0 Clear Clear Clear solution solutionsolution 21 Trifluoroethanol Heptane 50.0 50.0 Slight turbid Slighttuibid Slight Turbid 22 Trifluoroethanol TBME 50.0 50.0 Thick slurryThick Slurry precipitation 23 Trifluoroethanol Acetonitrile 50.0 50.0Thick Thick Slurry precipitation precipitation

TABLE 12 Results from Anti-solvent Addition Cooling/ Temperature CyclingCrystallizations Purity Solvent/ % by Sample solvent Anti- % anti- XRPDHPLC ID system solvent Solvent solvent analysis (%) 1 MethanolAcetonitrile 73.4 26.6 Crystalline 97.86 2 Methanol EA 73.7 26.3Crystalline 98.19 3 Methanol THF 73.7 26.3 Crystalline 96.43 4 MethanolAcetone 73.7 26.3 Crystalline 97.86 5 Methanol MEK 73.7 26.3 Crystalline97.88 6 Methanol Toluene 73.7 26.3 Crystalline 97.46 7 Methanol Heptane73.7 26.3 Crystalline 97.70 8 Ethanol EA 74.4 25.6 Poorly 98.01crystalline 9 Ethanol THF 74.4 25.6 Poorly 96.69 crystalline 10 EthanolAcetone 74.4 25.6 Partially 98.59 crystalline 11 Ethanol MEK 74.4 25.6Partially 98.31 crystalline 12 Ethanol Toluene 74.4 25.6 Partially 98.03crystalline 13 Ethanol Heptane 74.4 25.6 Partially 98.11 crystalline 14Ethanol TBME 74.4 25.6 Partially 97.87 crystalline 15 EthanolAcetonitrile 74.4 25.6 Partially 98.66 crystalline 16 Trifluoro- EA 50.050.0 Partially 97.10 ethanol crystalline 17 Trifluoro- THF 50.0 50.0Partially 97.41 ethanol crystallite 18 Trifluoro- Acetone 50.0 50.0Poorly 97.31 ethanol crystalline 19 Trifluoro- MEK 50.0 50.0 Poorly97.99 ethanol crystalline 20 Trifluoro- Toluene 50.0 50.0 ethanol 21Trifluoro- Heptane 50.0 50.0 ethanol 22 Trifluoro- TBME 50.0 50.0 Poorly97.71 ethanol crystalline 23 Trifluoro- Acetonitrile 50.0 50.0 Poorly97.47 ethanol crystalline

Anti-Solvent Addition Crystallizations

Further anti-solvent addition crystallizations were carried out usingmethanol and ethanol as the solvents with anti-solvents (75% v/v) addedat 50° C. and 5° C. The anti-solvents used were acetonitrile, ethylacetate, THF, acetone, MEK, toluene, TBME and heptane. The followingresults and observations were obtained from these experiments:

-   -   The wet material isolated from methanol/anti-solvent mixtures        was free flowing but the wet material from ethanol/anti-solvents        and trifluoroethanol/anti-solvents was gel like.    -   Observations and results are summarized in Table 13 and Table 14        respectively.    -   XRPD analysis on the isolated material revealed that crystalline        material was produced from methanol/anti-solvent mixtures except        from the methanol/toluene mixture at 50° C. where a partially        crystalline material was observed. Predominantly amorphous to        partially crystalline material was produced from        ethanol/anti-solvent mixtures.

TABLE 13 Observations from Cooling/Temperature Cycling Crystallizationsusing Solvent/Anti-solvent Mixtures Observation Observations Solvent/ %after Anti- at 5° C. (after Sample solvent Anti- Temper- % anti-Observation solvent temperature ID system solvent ature Solvent solventat 50° C. addition cycling) 1 Methanol Acetonitrile 50° C. 25 75 ClearClear solution Thick slurry solution followed by slow crystallization 2EA 25 75 Clear Clear solution Thick slurry solution followed by slowcrystallization 3 THF 25 75 Clear Clear solution Slurry (very solutionslow crystallization) 4 Acetone 25 75 Clear Clear solution Thick slurrysolution followed by slow crystallization 5 MEK 25 75 Clear Clearsolution Thick slurry solution followed by slow crystallization 6Toluene 25 75 Clear Clear solution Thick slurry solution followed byslow crystallization 7 TBME 25 75 Clear Clear solution Thick slurrysolution followed by slow crystallization 8 Ethanol Acetonitrile 50° C.25 75 Clear Clear solution Thick slurry solution (Gel like) 9 EA 25 75Clear Clear solution Thick slurry solution (Gel like) 10 THF 25 75 ClearClear solution Slurry (Gel solution like) 11 Acetone 25 75 Clear Clearsolution Thick slurry solution (Gel like) 12 MEK 25 75 Clear Clearsolution Pale yellow solution slurry (Gel like) 13 Toluene 25 75 ClearClear solution Thick slurry solution (Gel like) 14 TBME 25 75 ClearPrecipitation- Thick slurry solution Gel like (Gel like) 15 Heptane 2575 Clear Turbid Thick slurry solution (Gel like) 16 MethanolAcetonitrile  5° C. 25 75 Slurry Thick Thick slurry precipitation 17 EA25 75 Slurry Thick slurry Slurry 18 THF 25 75 Slurry Thick Slurryprecipitation 19 Acetone 25 75 Slurry Thick Slurry precipitation 20 MEK25 75 Slurry Thick Slurry precipitation 21 Toluene 25 75 Slurry Thickslurry Slurry 22 TBME 25 75 Slurry Thick slurry Slurry 23 EthanolAcetonitrile  5° C. 25 75 Turbid Thick Slurry precipitation 24 EA 25 75Slight Thick Slurry Turbid precipitation 25 THF 25 75 Slight Thick Thinslurry Turbid precipitation 26 Acetone 25 75 Slight Thick Thin slurryTurbid precipitation 27 MEK 25 75 Slight Thick Thin slurry Turbidprecipitation 28 Toluene 25 75 Slight Thick Very thin Turbidprecipitation slurry 29 TBME 25 75 Slight Thick Thin slurry Turbidprecipitation 30 Heptane 25 75 Slight Thick Thin slurry Turbidprecipitation

TABLE 14 Results from Cooling/Temperature Cycling Crystallizations usingSolvent/Anti-solvent Mixtures % Sample Anti- % anti- HPLC ID Solventsolvent Temperature Solvent solvent XRPD % 1 Methanol Acetonitrile 50°C. 25.0 75.0 Crystalline 96.94 2 EA 25.0 75.0 Crystalline 97.26 3 THF25.0 75.0 Crystalline 98.85 4 Acetone 25.0 75.0 Crystalline 97.49 5 MEK25.0 75.0 Crystallite 97.81 6 Toluene 25.0 75.0 Partially 98.01crystalline 7 TBME 25.0 75.0 Crystalline 97.35 8 Ethanol Acetonitrile50° C. 25.0 75.0 Crystalline 98.32 9 EA 25.0 75.0 Partially 96.76crystalline 10 THF 25.0 75.0 Poorly 95.94 crystalline 11 Acetone 25.075.0 Partially 98.87 Crystalline 12 MEK 25.0 75.0 Predominantly 97.49amorphous 13 Toluene 25.0 75.0 Predominantly 97.08 amorphous 14 TBME25.0 75.0 Partially 98.81 crystalline 15 Heptane 25.0 75.0 Predominantly97.44 amorphous 16 Methanol Acetonitrile  5° C. 25 75 Crystalline 97.617 EA 25 75 Crystalline 97.55 18 THF 25 75 Crystalline 97.57 19 Acetone25 75 Crystalline 97.55 20 MEK 25 75 Crystalline 97.42 21 Toluene 25 75Crystalline 97.30 22 TBME 25 75 Crystalline 97.12 23 EthanolAcetonitrile  5° C. 25 75 Partially 98.95 crystalline 24 EA 25 75Partially 98.02 crystalline 25 THF 25 75 Partially 97.85 crystalline 26Acetone 25 75 Partially 98.42 crystalline 27 MEK 25 75 Partially 98.52crystalline 28 Toluene 25 75 Predominantly 97.42 amorphous 29 TBME 25 75Partially 98.51 crystalline 30 Heptane 25 75 Partially 97.44 crystalline

Seeded Cooling Crystallization using Solvent/Anti-Solvent Mixtures

Crystalline Boc-D-Arg-DMT-Lys(Boc)-Phe-NH₂ was previously obtained frommethanol and methanol/anti-solvent mixtures by cooling/temperaturecycling crystallizations. The same crystalline form was observed and wasdesignated as Form 1. Seeded cooling crystallizations ofBoc-D-Arg-DMT-Lys(Boc)-Phe-NH₂ using solvent/anti-solvent mixtures werecarried out, where crystallizations were seeded using Form 1 at 50° C.followed by a slow cool to 5° C. The following results and observationswere obtained from these experiments:

-   -   After seeding with Form 1 at 50° C., seed persisted and further        nucleation was observed during granulation at 50° C. in every        experiment.    -   At 5° C., a thick slurry was observed.    -   The material isolated from methanol/anti-solvent mixtures was        free flowing. Ethanol/anti-solvents produced gel-like material        and after drying this material was observed to be partially        glass-like.    -   Observations and results are summarized in Table 15 and Table 16        respectively.    -   XRPD analysis on the isolated material revealed that crystalline        material was produced from methanol/anti-solvent mixtures.        Partially crystalline material was observed from        ethanol/anti-solvent mixtures

TABLE 15 Observations from Seeded Cooling Crystallizations usingSolvent/Anti-solvent Mixtures Observations Observations after % anti-after granulation Observations Anti- solvent/ Obser- Anti-solvent for 1hour at at Observations Sample solvent/ % co- vations addition at 50° C.(after 5° C. before ID Solvent co-solvent Solvent solvent at 50° C. 50°C. seeding) (cooling) isolation 1 Methanol Acetone 75 25 Clear ClearThin slurry- Thick slurry Slurry solution solution crystallization 2Methanol 2-propanol 75 25 Clear Clear Thin slurry- Thick slurry Slurrysolution solution crystallization 3 Methanol 2-butanol 75 25 Clear ClearThin slurry- Thick Slurry solution solution crystallizationprecipitation 4 Methanol TBME 75 25 Clear Clear Thin slurry- ThickSlurry solution solution crystallization precipitation 5 MethanolEthanol 50 50 Clear Clear Thin slurry- Thick slurry Slurry solutionsolution crystallization 6 Ethanol 2-propanol 75 25 Clear Clear Thinslurry- Thick Slurry solution solution crystallization precipitation 7Ethanol 2-butanol 75 25 Clear Clear Thin slurry- Thick Slurry solutionsolution crystallization precipitation

TABLE 16 Results from Seeded Cooling Crystallizations usingSolvent/Anti-solvent Mixtures Anti- % anti- Observations Samplesolvent/co- % solvent/co- before HPLC ID Solvent solvent Solvent solventIsolation XPRD (%) 1 Methanol Acetone 75 25 Slurry Crystalline 97.81 2Methanol 2-propanol 75 25 Slurry Crystalline 97.95 3 Methanol 2-butanol75 25 Slurry Crystalline 98.27 4 Methanol TBME 75 25 Slurry Crystalline98.27 5 Methanol Ethanol 50 50 Slurry Crystalline 97.69 6 Ethanol2-propanol 75 25 Slurry Partially 97.72 crystalline 7 Ethanol 2-butanol75 25 Slurry Partially 97.58 crystalline

Scale-Up Seeded Cooling Crystallizations Using Solvent/Anti-SolventMixtures

Crystallization scale-up experiments were carried out withBoc-D-Arg-DMT-Lys(Boc)-Phe-NH₂ on a ca. 250 mg scale, using methanol,methanol/acetonitrile, methanol/THF and ethanol/acetone solvent systems.The following results and observations were obtained from theseexperiments:

-   -   After seeding at 50° C., seed persisted and further nucleation        was observed during granulation at 50° C. for every experiment.    -   At 5° C., either a thick slurry or freely stirrable slurry was        observed.    -   The material isolated from methanol and methanol/anti-solvent        mixtures was free flowing, the ethanol/acetone solvent system        produced a gel-like material and after drying the material was        observed to be partially glass-like.    -   Observations and results are summarized in Table 17 and Table 18        respectively. Methanol (100 mg/mL)    -   XRPD analysis on the isolated material showed that crystalline        material was produced.    -   PLM analysis of the dried material indicated that the material        was birefringent with no well-defined morphology.    -   TG/DTA showed a weight loss of ca. 1.97% from the outset up to        ca. 169° C. An endothermic event was observed at an onset of        169.2° C. (peak at 178.7° C.) in the DTA.    -   The experiment produced crystalline material with a 85.06%        theoretical yield.    -   HPLC analysis on the dried material showed an uplift in purity        from 97.5% (purity of input material) to 98.5%.        Methanol: Acetonitrile (75:25% v/v)    -   XRPD analysis on the isolated material showed that crystalline        material was produced.    -   PLM analysis of the dried material indicated that the material        was birefringent with no well    -   defined morphology.    -   TG/DTA showed a weight loss of ca. 2.0% from the outset up to        ca. 170° C. An endothermic event was observed at an onset of ca.        170.9° C. (peak at 180° C.) in the DTA.    -   The experiment produced crystalline material with a 88.97%        theoretical yield.    -   HPLC analysis on the dried material showed an uplift in purity        from 97.5% (purity of input material) to 98.6%.

Methanol:THF (25:75% v/v)

-   -   XRPD analysis on the isolated material showed that crystalline        material was produced.    -   PLM analysis of the dried material indicated that the material        was birefringent with no well-defined morphology.    -   TG/DTA showed a weight loss of ca. 1.79% from the outset up to        ca. 165° C. An endothermic event was observed at an onset of        165.2° C. (peak at 173.1° C.) in the DTA.    -   The experiment produced crystalline material with a 31.57%        theoretical yield.    -   HPLC analysis on the dried material showed an uplift in purity        from 97.5% (purity of input material) to 99.2%.

Ethanol: Acetone (25:75% v/v)

-   -   XRPD analysis on the isolated material showed that predominantly        amorphous material was produced.    -   PLM analysis of the dried material indicated that the material        was non-birefringent.    -   TG/DTA showed a weight loss of ca. 1.3% from the outset up to        ca. 144° C. An endothermic event was observed at an onset of        144.6° C. (peak at 153.4° C.) in the DTA. TG/DTA was observed to        be similar to input Boc-D-Arg-DMT-Lys(Boc)-Phe-NH₂,        predominantly amorphous material.    -   The experiment produced predominantly amorphous material with a        84.39% theoretical yield.    -   HPLC analysis on the dried material showed an uplift in purity        from 97.5% (purity of input material) to 98.4%.

TABLE 17 Observations from Scale-up Seeded Cooling CrystallizationsObservations Observation Observations after at 50° C. after granulationbefore addition of for 1 hour at Sample Anti- Anti- addition of antisolvent 50° C. (after Observations ID Solvent solvent Solvent solventanti-solvent at 50° C. seeding) at 5° C. 1 Methanol 100.00 Clear N/AThin slurry- Thick Slurry solution crystallization 2 MethanolAcetonitrile 75.00 25.00 Clear Clear solution Thin slurry- Thick Slurrysolution crystallization 3 Methanol THF 25.00 75.00 Clear Clear solutionThin slurry- Slurry solution crystallization 4 Ethanol Acetone 25.0075.00 Clear Clear solution Thin slurry- Slurry (Gel- solutioncrystallization like material)

TABLE 18 Results from Scale-up Seeded Cooling Crystallizations Mean MeanPurity % Conc. In Solid Area Mother Sample Anti- Purity (Mother LiquorTheoretical ID Solvent solvent XRPD % Area Liquor) mg/mL yield (%) 1Methanol Crystalline 98.51 97.69 94.94 85.06 2 Methanol AcetonitrileCrystalline 98.63 97.66 91.93 88.97 3 Methanol THF Crystalline 99.2498.98 38.97 31.57 4 Ethanol Acetone Predominantly 98.44 96.48 10.3884.39 amorphous

Primary Salt Screening

A limited salt screen was carried out on Boc-D-Arg-DMT-Lys(Boc)-Phe-NH₂with the aim of locating crystalline salts in order to assess thepotential for purification through salt formation.

Salt screening on Boc-D-Arg-DMT-Lys(Boc)-Phe-NH₂ was carried out usinghydrochloric acid, p-toluenesulfonic acid, methane sulfonic acid, oxalicacid, L-tartaric acid, fumaric acid, benzoic acid and succinic acidusing acetone, acetonitrile:water (50:50% v/v), ethanol and methanol assolvent systems. The following results and observations were obtainedfrom these experiments:

Salt Formation Using Hydrochloric Acid

-   -   After temperature cycling, gel-like material was observed when        using acetone, acetonitrile:water (50:50% v/v) and ethanol. A        slurry was observed when using methanol as the solvent.    -   XRPD analysis on the isolated material showed that crystalline        material (Form 1, free base) was produced from methanol and        predominantly amorphous material was produced from the acetone,        acetonitrile: water (50:50% v/v) and ethanol solvent systems.

Salt Formation Using p-Toluene Sulfonic Acid

-   -   After temperature cycling, a clear solution with some solids at        the bottom of the vial was observed using acetone as the        solvent. Gel-like material was observed using acetonitrile:water        (50:50% v/v), a slurry was observed using methanol and a clear        solution was produced using ethanol.    -   XRPD analysis on the isolated material showed that partially        crystalline material having an XRPD pattern different from the        free base Form 1 was produced from acetone and acetonitrile:        water (50:50% v/v). Partially crystalline material, having some        peaks in common with the free base Form 1 was observed from        methanol.

Salt Formation Using Methane Sulfonic Acid

-   -   After temperature cycling, a slurry was observed using acetone        and ethanol, gel-like material was observed using acetonitrile:        water (50:50% v/v) and a clear solution was produced from        methanol as the solvent.    -   XRPD analysis on the isolated material showed that partially        crystalline material having an XRPD pattern different from free        base Form 1 was produced from acetone and acetonitrile:water        (50:50% v/v). Predominantly amorphous material was observed from        ethanol.

Salt Formation Using Oxalic Acid

-   -   After temperature cycling, a gel-like material was observed        using acetonitrile: water (50:50% v/v) and ethanol. A slurry was        observed when using acetone and methanol as the solvents.    -   XRPD analysis on the isolated material revealed that crystalline        material (Free base, Form 1) was produced from methanol,        predominantly amorphous material was produced from ethanol and a        partially crystalline material having an XRPD pattern different        from free base Form 1 was produced from acetonitrile: water        (50:50% v/v). Crystalline material having an XRPD pattern        different from free base Form 1 was observed from acetone.

Salt Formation Using L-Tartaric Acid

-   -   After temperature cycling, a slurry was observed when using        acetone and methanol, whilst a gel-like material was observed        when using acetonitrile: water (50:50% v/v) and ethanol.    -   XRPD analysis on the isolated material revealed that crystalline        material (Free base, Form 1) was produced from methanol and        predominantly amorphous material was produced form acetone and        ethanol. Partially crystalline material having an XRPD pattern        different from free base Form 1 was produced from acetonitrile:        water (50:50% v/v).

Salt Formation Using Fumaric Acid

-   -   After temperature cycling, a slurry was observed when using        acetone and methanol, whilst a gel-like material was observed        when using acetonitrile: water (50:50% v/v) and ethanol.    -   XRPD analysis on the isolated material showed that crystalline        material (Free base, Form 1) was produced from methanol and        predominantly amorphous material was produced form acetone and        ethanol. Partially crystalline material having an XRPD pattern        different from the free base Form 1 was produced from        acetonitrile: water (50:50% v/v).

Salt Formation Using Benzoic Acid

-   -   After temperature cycling, gel-like material was observed using        acetonitrile: water (50:50% v/v) and ethanol. A slurry was        observed when using acetone as the solvent. Solid was observed        from methanol.    -   XRPD analysis on the isolated material revealed that poorly        crystalline material (Free base, Form 1) was produced from        acetone and ethanol. Crystalline material (slightly different        from free base Form 1) was produced from acetonitrile: water        (50:50% v/v) and methanol.

Salt Formation Using Succinic Acid

-   -   After temperature cycling, a gel-like material was observed when        using acetonitrile: water (50:50% v/v) and a slurry was observed        when using ethanol or methanol as the solvent.    -   Solid was observed from acetone.    -   XRPD analysis on the isolated material showed that partially        crystalline material (Free base, Form 1) was produced from        acetone, ethanol and methanol. Poorly crystalline material        (slightly different from free base Form 1) was produced from        acetonitrile: water (50:50% v/v).        Observations are summarized in Table 19 and Table 20 and results        are summarized in Table 21 and Table 22.

TABLE 19 Observations from Primary Salt Screening ObservationObservation before after Observation Observation addition of addition ofafter after Solvent/ Initial counterion counterion temperaturetemperature Sample solvent observations solution/slurry solution atcycle (16 cycling (40 ID system Counterion at 50° C. at 50° C. 50° C.hours) hours) 1 Acetone HCl Slurry Slurry Slurry Gel like Gel like 2Acetonitrile: Clear Clear solution Slurry Gel like Gel like watersolution (50:50% v/v) followed by precipitation: slurry 3 Ethanol ClearTurbid Clear Gel like Gel like solution solution followed byprecipitation: slurry 4 Methanol Clear Slurry Slurry Slurry Slurrysolution followed by precipitation: slurry 5 Acetone P-toluene SlurrySlurry Clear Clear Clear sulfonic solution solution/ solution/ acid somesolids some solids at the bottom at the bottom 6 Acetonitrile: ClearClear solution Turbid Gel like Gel like water solution (50:50% v/v)followed by precipitation: slurry 7 Ethanol Clear Slurry Clear ClearClear solution solution solution solution followed by precipitation:slurry 8 Methanol Clear Slurry Slurry Slurry Slurry solution followed byprecipitation: slurry 9 Acetone Methane Slurry Slurry Slurry SlurrySlurry 10 Acetonitrile: sulfonic Clear Slurry Clear Gel like Gel likewater acid solution solution (50:50% v/v) followed by precipitation:slurry 11 Ethanol Clear Turbid Slight Slurry Slurry solution Turbidfollowed by precipitation: slurry 12 Methanol Clear Slurry Clear ClearClear solution solution solution solution followed by precipitation:slurry 13 Acetone Oxalic Acid Slurry Slurry Slurry Slurry Slurry 14Acetonitrile: Clear Clear solution Clear Gel like Gel like watersolution solution (50:50% v/v) followed by precipitation: slurry 15Ethanol Clear Turbid Slurry Gel like Gel like solution followed byprecipitation: slurry 16 Methanol Clear Slurry Slurry Slurry Slurrysolution followed by precipitation: slurry

TABLE 20 Observations from Primary Salt Screening Observation beforeObservation addition of after Observation Observation counterionaddition of after after Solvent/ Initial solution/ counteriontemperature temperature Sample solvent observations slurry at solutionat cycle (16 cycling (40 ID system Counterion at 50° C. 50° C. 50° C.hours) hours) 17 Acetone L-Tartaric Slurry Slurry Slurry Slurry Slurry18 Acetonitrile: Acid Clear Slurry Slurry Gel like Gel like watersolution (50:50% v/v) followed by precipitation: slurry 19 Ethanol ClearSlurry Slurry Gel like Gel like solution followed by precipitation:slurry 20 Methanol Clear Slurry Slurry Slurry Slurry solution followedby precipitation: slurry 21 Acetone Fumaric Slurry Slurry Slurry SlurrySlurry 22 Acetonitrile: Acid Clear Clear Clear Gel like Gel like watersolution solution solution (50:50% v/v) followed by precipitation:slurry 23 Ethanol Clear Turbid Clear Gel like Gel like solution solutionfollowed by precipitation: slurry 24 Methanol Clear Slurry Slurry SlurrySlurry solution followed by precipitation: slurry 25 Acetone BenzoicSlurry Slurry Slurry Slurry Slurry 26 Acetonitrile: Acid Clear ClearClear Gel like Gel like water solution solution solution (50:50% v/v)followed by precipitation: slurry 27 Ethanol Clear Turbid Clear Gel likeGel like solution solution followed by precipitation: slurry 28 MethanolClear Slurry Slurry Solid Dry solid solution followed by precipitation:slurry 29 Acetone Succinic Slurry Slurry Slurry Solid Dry solid 30Acetonitrile: Acid Clear Turbid Clear Gel like Gel like water solutionsolution (50:50% v/v) followed by precipitation: slurry 31 Ethanol ClearTurbid Slurry Slurry Slurry solution followed by precipitation: slurry32 Methanol Clear Slurry Slurry Slurry Slurry solution followed byprecipitation: slurry

TABLE 21 Results from Primary Salt Screening Sample Solvent/ Counter- %Purity ID Solvent system ion XRPD by HPLC 1 Acetone HCl PredominantlyAmorphous (Same as input material) 2 Acetonitrile: Poorly Crystalline97.94 water (slightly different than (50:50% v/v) input material) 3Ethanol Predominantly Amorphous (Same as input material) 4 MethanolCrystalline, same form as crystalline free material 5 Acetone P-Partially crystalline 99.25 toluene (different form from sulfoniccrystalline free material) 6 Acetonitrile: acid Poorly Crystalline 89.97water (slightly different than (50:50% v/v) input material) 7 Ethanol 8Methanol Partially crystalline (some peaks matching with the crystallinefree material) 9 Acetone Methane Partially crystalline 84.44 sulfonic(different form from acid crystalline free material) 10 Acetonitrile:Poorly Crystalline water (slightly different than (50:50% v/v) inputmaterial) 11 Ethanol Predominantly Amorphous (slightly different thaninput material) 12 Methanol 13 Acetone Oxalic Crystalline (different98.02 Acid form from crystalline free material) 14 Acetonitrile: PoorlyCrystalline 87.29 water (slightly different than (50:50% v/v) inputmaterial) 15 Ethanol Predominantly Amorphous (Same as input material) 16Methanol Crystalline, same form as crystalline free material

TABLE 22 Results from Primary Salt Screening Sample Solvent/Solvent %Purity by ID system Counterion XRPD HPLC 17 Acetone L-TartaricPredominantly amorphous acid (slightly different than input material) 18Acetonitrile:water Poorly crystalline (slightly 98.64 (50:50% v/v)different than input material) 19 Ethanol Poorly crystalline (slightlydifferent than input material) 20 Methanol Crystalline, same form ascrystalline free material 21 Acetone Fumaric Predominantly amorphous(same acid as input material) 22 Acetonitrile:water Poorly crystalline(slightly 98.65 (50:50% v/v) different than input material) 23 EthanolPredominantly amorphous (mixture of input free material and crystallinefree material) 24 Methanol Crystalline, same form as crystalline freematerial 25 Acetone Benzoic Poorly crystalline (similar to acidcrystalline free material) 26 Acetonitrile:water Crystalline (slightlydifferent from 98.71 (50:50% v/v) crystalline free material) 27 EthanolPoorly crystalline, similar form as crystalline free material) 28Methanol Crystalline (slightly different than 98.97 crystalline freematerial) 29 Acetone Succinic Partially crystalline (mixture of acidinput free material and crystalline free material) 30 Acetonitrile:waterPoorly crystalline (slightly 98.68 (50:50% v/v) different than inputmaterial) 31 Ethanol Partially crystalline, same form as crystallinefree material 32 Methanol Partially crystalline, same form ascrystalline free material

Summary of Results

Initial characterization of Boc-D-Arg-DMT-Lys (Boc)-Phe-NH₂, showed itto be predominantly amorphous by XRPD analysis and non-birefringent byPLManalysis, exhibiting no clearly defined morphology. TG/DT analysisshowed a weight loss of ca. 2.79% from the outset up to ca. 144° C.,followed by a weight loss of ca. 0.72%, associated with an endothermicevent at ca. 144.3° C. (onset ca. 155.2° C.). DSC analysis showed abroad endothermic event from the outset up to ca. 140° C. with a furtherendothermic event observed at ca. 155.6° C. (onset at ca. 140.2° C.). KFanalysis indicated a water content of ca. 3.61%, while GVS analysisindicated the material was highly hygroscopic, with a mass increase ofca. 10% from 40-90% RH. The purity of the received material was 97.50%by HPLC.

An approximate solvent solubility screen was carried out using 19solvent systems and yielded a range of solubilities. The receivedmaterial was found to be highly soluble, with methanol,trifluoroethanol, acetonitrile:water (50:50% v/v) and DMSO:acetone(50:50% v/v) giving solubility values of ≥200 mg/mL. A solubility of ca.140 mg/mL was obtained in ethanol with a ca. 100 mg/mL solubilityobserved in 2-propanol:water (50:50% v/v) and ethanol:water (50:50%v/v). Moderate solubility (ca. 58 to 24 mg/mL) was observed inacetone:water (50:50 v/v), methanol:water (50:50 v/v), and DMSO:water(50:50 v/v), with poor solubility (<17 mg/mL) obtained in all othersolvent systems investigated, including acetone, dichloromethane,2-butanol, 2-propanol, methyl ethyl ketone, toluene, THF, ethyl acetateand acetonitrile. The residual solids from some of the solvent systemsshowing poor solubility were analyzed by XRPD after slurrying at 50° C.overnight, but all diffractograms indicated that the material remainedpredominantly amorphous.

Small-scale crystallization screening experiments were carried outinvestigating cooling, temperature cycling, anti-solvent addition andseeding techniques. Cooling followed by temperature cyclingcrystallizations were carried out using seven different initial solventmixtures. The material was dissolved in ethanol, methanol,trifluoroethanol, acetone:water (50:50% v/v, Acetonitrile:water (50:50%v/v) and DMSO:water (80:20% v/v) at 50° C. The solutions were cooleddown to 5° C. then temperature cycled between 40° C. and 5° C.Crystallization of material at 50° C. was observed with methanol at boththe process concentrations of 150 mg/mL and 100 mg/mL and crystallinematerial was isolated (Form 1) which was birefringent by PLM analysis,with no defined morphology. The purity of the solid isolated frommethanol was 98.24% (ca. 150 mg/mL) and 98.62% (ca. 100 mg/mL),indicating that crystallization offered purity uplift over the inputpredominantly amorphous material. Predominantly amorphous solids wereisolated from ethanol and acetone: water (50:50% v/v) showing purityvalues of 98.80% and 98.81%, indicating a purity uplift over the inputmaterial, however the wet material isolated from these solvent systemswas gel-like. The other solvent systems did not yield solids.

Anti-solvent addition followed by cooling/temperature cyclingcrystallizations were carried out using 23 solvent/anti-solventmixtures. The material was dissolved in ethanol, methanol, andtrifluoroethanol at 50° C. Anti-solvents acetonitrile, ethyl acetate,THF, acetone, MEK, toluene, heptane and TBME (for ethanol andtrifluoroethanol) were added at 50° C. to achieve ratios ofmethanol/anti-solvent (73.7:26.3% v/v), ethanol/anti-solvent (74.4:25.6%v/v) and trifluoroethanol/anti-solvent (50:50% v/v). Clear solutionswere observed at 50° C. with methanol/anti-solvents but forethanol/anti-solvent mixtures and trifluoroethanol/anti-solvent mixturesfor most experiments turbidity to thick precipitation was observed. Thecrystallizations were cooled down to 5° C. then temperature cycledbetween 40° C. and 5° C. Crystalline material was isolated frommethanol/antisolvent mixtures (Form 1). The purity values of the solidsisolated from methanol/anti-solvent mixtures were between 96.43% and98.19%, indicating that the crystallization offered a purity uplift forsome of the methanol/anti-solvent mixtures. Poorly crystalline topartially crystalline solids were isolated from ethanol/anti-solventmixtures and trifluoroethanol/anti-solvent mixtures. Purity of thesolids was between 96.69% and 98.66%, but the wet material isolated fromthese solvent systems was gel-like.

Further anti-solvent addition crystallizations were carried out using 15solvent/anti-solvent mixtures. The material was dissolved in methanoland ethanol at 50° C. Anti-solvents acetonitrile, ethyl acetate, THF,acetone, MEK, toluene, TBME and heptane (for ethanol) were added at 50°C. and 5° C. to achieve solvent/anti-solvent ratios of (25:75% v/v).Crystalline material was isolated from methanol/anti-solvent mixturesexcept from the methanol/toluene mixture at 50° C., where partiallycrystalline material was observed. The maximum uplift of purity wasobserved from methanol/THF (THF addition at 50° C.) where a purity of98.85% was afforded. Predominantly amorphous to partially crystallinematerial was produced from ethanol/anti-solvent mixtures with a maximumuplift of purity (98.95%) observed from ethanol/acetonitrile(acetonitrile addition at 5° C.). The wet material isolated fromethanol/anti-solvent mixtures was however gel-like.

Seeded anti-solvent addition crystallizations using solvent/anti-solventmixtures were carried out in 7 solvent systems. Formethanol/anti-solvent mixtures (75:25% v/v), acetone, 2-propanol,2-butanol, TBME were used as the anti-solvents and a methanol/ethanol(50:50% v/v) ratio was also used. For ethanol/anti-solvent mixtures(75:25% v/v), 2 propanol and 2-butanol were used as the anti-solvents.The material was dissolved in methanol and ethanol at 50° C.Anti-solvents were added at 50° C., affording clear solutions. The clearsolutions were seeded with Form 1 at 50° C. followed by granulation for1 hour where crystallization was observed. The crystallizations werecooled down to 5° C. Crystalline material was isolated frommethanol/anti-solvent mixtures and partially crystalline material fromethanol/anti-solvent mixtures. The maximum uplift in purity was observedusing methanol/2-butanol and methanol/TBME, showing a purity of 98.27%for both solvent systems.

The seeded anti-solvent addition cooling crystallizations were furtherscaled up to 250 mg scale using methanol (100 mg/mL),methanol/acetonitrile (75:25% v/v), methanol/THF (25:75% v/v) andethanol/acetone (25:75% v/v) solvent systems. Crystalline material wasisolated from methanol and methanol/anti-solvent mixtures andpredominantly amorphous material from the ethanol/acetone system. Thebest result was obtained from methanol/acetonitrile with a theoreticalyield of 88.97% and a purity of 98.63%. A purity of 99.24% was obtainedfrom methanol/THF (25:75% v/v), but the theoretical yield was only 31%.The predominantly amorphous material obtained from ethanol/acetone(25:75% v/v) showed a purity of 98.44% with a theoretical yield of84.39%.

A limited salt screen was carried out on Boc-D-Arg-DMT-Lys(Boc)-Phe-NH₂with the aim of locating a crystalline salt and assessing potentialpurification through salt formation. The counter ions and solventsystems used for the salt screening included hydrochloric acid,p-toluenesulfonic acid, methane sulfonic acid, oxalic acid, L-tartaricacid fumaric acid, benzoic acid and succinic acid in acetone,acetonitrile:water (50:50% v/v), ethanol and methanol. The material wasslurried/dissolved in the solvent systems at 50° C. In acetone, slurrieswere observed but in the remaining systems, clear solutions wereobtained. Within 1 hour of granulation at 50° C., crystallization ofmaterial was observed and a slurry was present for every experiment. Theexperiments were further diluted with the respective solvent system todissolve the crystallized material or to afford a stirrable slurry. Thecounterion solutions were added to the respective experiments at 50° C.The experiments were stirred at 50° C. and cooled down to 5° C. and thentemperature cycled between 5° C. to 40° C. for ca. 40 hours.

Salt screening using Hydrochloric acid produced predominantly amorphousto poorly crystalline gel-like material, having a diffractogram slightlydifferent from the input material in acetone, acetonitrile: water(50:50% v/v) and ethanol. Crystalline material (Forml, free base) wasobserved using methanol as the solvent.

Salt screening using p-Toluene sulfonic acid produced partiallycrystalline material having an XRPD pattern different from the free baseForm 1 in acetone. ¹H NMR analysis on the solids revealed salt formationand a purity of 99.25% was observed. Poorly crystalline gel-likematerial having a diffractogram slightly different from the inputmaterial was observed from acetonitrile:water (50:50% v/v) with a purityof 89.97%. Methanol produced partially crystalline material similar tothe crystalline free base.

Salt screening using Methane sulfonic acid produced partiallycrystalline material having an XRPD pattern different from the free baseForm 1 in acetone with a purity of 84.44%. Poorly crystalline andpredominantly amorphous gel-like material having a diffractogramslightly different from the input material was observed fromacetonitrile: water (50:50% v/v) and ethanol respectively.

Salt screening using Oxalic acid produced crystalline material having aXRPD pattern different from the crystalline free base Form 1 in acetone,with a purity of 98.02%. ¹H NMR analysis on the solids indicated saltformation. Poorly crystalline gel-like material having a diffractogramslightly different from the input material was observed fromacetonitrile: water (50:50% v/v) with a purity of 87.29%. No saltformation was observed using ethanol and methanol as solvents as thesesystems produced material having diffractograms identical to the inputmaterial and crystalline free base respectively.

Salt screening using L-Tartaric acid produced predominantly amorphousand poorly crystalline material having an XRPD pattern slightlydifferent from the input material in acetone, acetonitrile:water (50:50%v/v) and ethanol respectively. Poorly crystalline material produced fromacetonitrile:water (50:50% v/v) showed a purity of 98.64%. Crystallinefree base Form 1 was produced from methanol.

Salt screening using Fumaric acid produced predominantly amorphousmaterial having an XRPD pattern similar to the amorphous input and apattern showing a mixture of crystalline and amorphous free base fromacetone and ethanol respectively. Poorly crystalline material producedfrom acetonitrile: water (50:50% v/v) showed a purity of 98.65%.Crystalline free base Form 1 was produced from methanol.

Salt screening using Benzoic acid produced poorly crystalline materialhaving an XRPD pattern similar to crystalline free base Form 1 fromacetone and ethanol. Crystalline material having an XRPD patternslightly different from crystalline free base Form 1 was produced fromacetonitrile:water (50:50% v/v) and methanol with purities of 98.71% and98.87% respectively.

Salt screening using Succinic acid produced partially crystallinematerial similar to crystalline form 1 was observed from acetone,ethanol and methanol. Poorly crystalline material produced fromacetonitrile: water (50:50% v/v) showed a purity of 98.68%.

Overall, the crystallization screening study on Boc-D-Arg-DMT-Lys(Boc)-Phe-NH₂ indicated that crystalline material could be obtained byre-crystallization of the predominantly amorphous solid in methanol andmethanol/anti-solvent mixtures. An uplift in purity was observed throughcrystallization of the intermediate. The primary salt screening study onBoc-D-Arg-DMT-Lys (Boc)-Phe-NH₂ resulted in crystalline material fromoxalic acid in acetone having a different XRPD pattern compared withfree base Form 1. Partially crystalline material having an XRPD patterndifferent from the free base Form 1 was obtained from p-toluene sulfonicacid in acetone. Further work would be required in order to betterascertain the nature of these solid forms

Overall, crystalline material could be obtained by re-crystallization ofthe predominantly amorphous solid in methanol and inmethanol/anti-solvent mixtures. The crystalline material wassuccessfully produced at a 250 mg scale using methanol (100 mg/mL),methanol/acetonitrile (75:25% v/v) and methanol/THF (25:75% v/v). Usingmethanol/acetonitrile (75:25% v/v) we obtained a yield of 88.97% andpurity of 98.63%. Limited salt screening resulted in crystalline andpartially crystalline material when using oxalic acid andp-Toluenesulfonic acid in acetone, respectively. Both of the materialsshowed XRPD diffractgrams different from the crystalline free baseForm 1. These salt formations also offered purity uplifts over the inputmaterial with a purity of 98.02% from the oxalic acid experiment and99.25% from the p-Toluenesulfonic acid experiment.

The approximate solvent solubility screen utilized nineteen solventsystems and yielded a range of solubilities. The received material wasfound to be highly soluble, with methanol, trifluoroethanol,acetonitrile:water (50:50% v/v) and DMSO:acetone (50:50% v/v) givingsolubility values of ≥200 mg/mL. A solubility of ca. 140 mg/mL wasobtained in ethanol with a ca. 100 mg/mL solubility observed in2-propanol:water (50:50% v/v) and ethanol:water (50:50% v/v). Moderatesolubility (ca. 58 to 24 mg/mL) was obtained in acetone:water (50:50v/v), methanol:water (50:50 v/v) and DMSO:water (50:50 v/v), with poorsolubility (≤17 mg/mL) obtained in all other solvent systemsinvestigated. The screen identified acetone, dichloromethane, 2-butanol,2-propanol, methyl ethyl ketone, toluene, THF, ethyl acetate andacetonitrile as potential anti-solvents.

Small-scale crystallization screening experiments were carried outinvestigating cooling, temperature cycling, anti-solvent addition andseeding techniques. Cooling/temperature cycling crystallizations usingmethanol (at process concentrations of 150 mg/mL and 100 mg/mL) yieldedcrystalline material which were birefringent by PLM analysis, with nodefined morphology. The purity of the crystallized solids isolated frommethanol was 98.24% (ca. 150 mg/mL) and 98.62% (ca. 100 mg/mL),indicating that crystallization offered a purity uplift over the inputpredominantly amorphous material (97.5%). Crystallized wet material fromacetone:water (50:50% v/v) and ethanol was gel-like and after drying,glass-like material was observed. Using water as part of thecrystallization solvent was observed to be unsuitable for production ofcrystalline material, but ethanol was further investigated. Anti-solventaddition/cooling/temperature cycling crystallizations usingmethanol/anti-solvents (73:27% v.v) also produced crystalline material(Form 1). Ethanol/anti-solvents (74:46% v/v) andtrifluoroethanol/anti-solvents (50:50% v/v) produced gel-like materialwhich was observed to be poorly crystalline to partially crystalline,but an uplift in purity over the input material was observed with someof these solvent systems. Anti-solvent addition crystallizations, whereanti-solvents (75% v/v) were added at 50° C. and 5° C. producedcrystalline material using methanol/anti-solvent mixtures at bothtemperatures. Ethanol/anti-solvent (25:75% v/v) mixtures again producedgel-like material which dried to a glass-like solid. Seeded coolingcrystallizations resulted in crystalline material frommethanol/anti-solvent mixtures and partially crystalline material fromethanol/anti-solvent mixtures. When ethanol was used along with methanol(50:50% v/v), crystalline material was observed, however the use ofethanol along with other solvents did not allow for crystallization. Anuplift in purity over the input material was observed with a maximumuplift of purity using methanol/2-butanol and methanol/TBME, where bothexperiments showed purities of 98.27%.

The seeded anti-solvent addition cooling crystallizations were furtherscaled up to 250 mg scale using methanol (100 mg/mL),methanol/Acetonitrile (75:25% v/v), methanol/THF (25:75% v/v) andethanol/acetone (25:75% v/v). Crystalline material was isolated frommethanol and methanol/anti-solvent mixtures and predominantly amorphousmaterial from ethanol/acetone system. Using methanol/acetonitrile weobtained a yield of 88.97% and purity of 98.63%. A purity of 99.24% wasobtained from methanol/THF (25:75% v/v), but the yield was only 31%. Thepredominantly amorphous material obtained from ethanol/acetone (25:75%v/v) showed a purity of 98.44% with a yield of 84.39%.

Limited salt screening was carried out onBoc-D-Arg-DMT-Lys(Boc)-Phe-NH₂, with the aim of locating a crystallinesalt and assessing potential purification through salt formation. Thescreen entailed the use of hydrochloric acid, p-toluenesulfonic acid,methane sulfonic acid, oxalic acid, L-tartaric acid, fumaric acid,benzoic acid and succinic acid as the counterions in acetone,acetonitrile:water (50:50% v/v), ethanol and methanol solvent systems.Limited success was seen from the salt screen with crystalline materialproduced from oxalic acid in acetone and partially crystalline materialfrom p-Toluene sulfonic acid in acetone, both having different XRPDpatterns compared with the crystalline free base Form 1. All other saltformation reactions resulted in predominantly amorphous, poorlycrystalline, partially crystalline and crystalline material having XRPDpatterns similar to or only slightly different from the amorphousreceived material or crystalline free base.

Crystallization of the Boc-D-Arg-DMT-Lys(Boc)-Phe-NH₂ intermediatematerial did not show an improvement in terms of hygroscopicity, but itdid allow for purification.

Example 3. HCl-IPA Deprotection of Boc-D-Arg-DMT-Lys(Boc)-Phe-NH₂

Equipment: A 1 L 3-neck round-bottomed flask equipped with a mechanicalstirrer, thermometer, addition funnel and a nitrogen inlet.

Procedures:

-   -   Charge Boc-D-Arg-DMT-Lys(Boc)-Phe-NH₂ (35.4 g. 0.042 mol, 1.0        eq).    -   Charge IPA (280 mL, 8 parts) and begin agitation.    -   Adjust temperature to 22° C. (19-25° C.).    -   Charge 5-6 M HCl in IPA (77 mL, 0.42 mol, 10.0 eq) over a period        of 10 minutes while maintaining temperature below 25° C.    -   Adjust temperature to 40-45° C.    -   Agitate mixture at 40-45° C. for a period of 1-2 h.    -   A sample of the mixture (ca. 0.5 mL) is removed for IPC#3        testing. Analytical methods and typical results are provided in        the appropriate sections below.    -   IPC#3 is used to determine reaction completion as indicated by        the disappearance of Boc-D-Arg-DMT-Lys(Boc)-Phe-NH₂ by UPLC.        Limit: Report only, % a/a.    -   Typical results for IPC#3: Boc-D-Arg-DMT-Lys(Boc)-Phe-NH₂ wrt        (Boc-D-Arg-DMT-Lys(Boc)-Phe-NH₂+D-Arg-DMT-Lys-Phe-NH₂)=ND.    -   Cool suspension to 22° C. (19-25° C.) over a period of 1-2 h.    -   Filter suspension.    -   Wash solids with IPA (3×70 mL, 3×2 parts).    -   Dry on filter under a stream of N₂ at room temperature for a        minimum of 17 h to give a white solid.    -   Unload the filter and weigh D-Arg-DMT-Lys-Phe-NH₂ solid.    -   Typical mass is 30.7 g, uncorrected for solvent.    -   A sample of the solid (ca. 250 mg) is removed for IPC#4 testing.        Analytical methods and typical results are provided in the        appropriate sections below.    -   IPC#4a is used to determine residual IPA by GC. Limit: Report        only, ppm.    -   Typical result for IPC#5a: IPA=4.4% w/w.    -   IPC#4b is used to determine purity of C745 by HPLC. Limit:        Report only, % a/a.    -   Typical result for IPC#5b: C745 purity=98.41% a/a.    -   Charge crude C789 to the reactor (30.7 g, 1.0 eq).    -   Charge MTBE (460 mL, 15 parts wrt crude C745) and begin        agitation.    -   Charge EtOH (92 mL, 3 parts).    -   Adjust temperature to reflux (ca. 55° C.).    -   Agitate suspension at reflux temperature for a period of 16-18        h.    -   Adjust temperature to 22° C. (19-25° C.) over 1-2 h.    -   Agitate suspension at 22° C. (19-25° C.) for a period of 2-3 h.    -   Filter suspension.    -   Wash solids with MTBE (2×60 mL, 2×2 parts).    -   Dry on filter under a stream of N₂ at room temperature for a        minimum of 12 h to give a white solid.

This initial process using 5-6M HCl in IPA resulted in the formation ofisopropyl ester analog as well as several t-butylated analogs asimpurities.

This solid required treatment with EtOH-MTBE to remove the IPA that wastrapped in the solid (presumably a solvate as drying at 100° C. forextended periods of time did reduce the level below a certain amount.

Although effective at removing the iPrOH and at reducing the iPr estercontent, it resulted in the formation of the ethyl ester.

Example 4. Improved Deprotection of Boc-D-Arg-DMT-Lys(Boc)-Phe-NH₂

A second procedure was developed using HCl in TFE with TIPS as a t-butylcation scavenger. This procedure avoids the formation of alkyl estersbut additionally reduced the number and amount of t-butylated analogspresent.

To a cooled (0-5° C.) slurry of Boc-D-Arg-DMT-Lys(Boc)-Phe-NH₂ (0.500 g,0.570 mmol) and triisopropylsilane (0.584 mL, 2.85 mmol) in2,2,2-trifluoroethanol (5.0 mL, 69 mmol) was added conc. hydrogenchloride (0.238 mL, 2.85 mmol) dropwise over approx. 5 min. After 10min, the ice bath was removed and the mixture stirred at ambienttemperature. After approx. 20 min at ambient temperature, all solids haddissolved leading to the formation of a biphasic mixture. After 1 h atambient temperature HPLC analysis showed the consumption ofBoc-D-Arg-DMT-Lys(Boc)-Phe-NH₂. The product purity was observed to be98.13 area % [Agilent 1100 HPLC, Waters XSelect CSH C18, 150×4.6 mm, 3.5micron, 1.0 ml/min, UV220 nm, Column temperature 30° C.; Solvent A: H2O(0.05% TFA); Solvent B: acetonitrile (0.05% TFA). Hold 1 min 95% A, 15min gradient 95% to 80% A, 5 min 80% to 50% A, 5 min 50% to 10% A, hold2 min at 10% A, 0.1 min gradient 10% to 95% A. Hold at 95% A forremainder of 36 min run time. Diluent 9:1 water/ACN]. An HPLC peak at aRRT of 1.04 was observed (0.9 area %). No TFE ester was observed byLCMS. After 90 min, the mixture was diluted with MeOAc (5 mL) affordinga white precipitate. Volatiles were removed at reduced pressure and thesolid concentrated from MeOAc (5 mL) to afford a free flowing whitesolid that was dried in vacuo overnight. Residual solvents observed by¹H NMR included TFE (7% w/w) and MeOAc (0.3% w/w). Product purity was98.08 area % as assayed by HPLC. The solid was slurried in MTBE/MeOAc(2:1, 10 ml) for 10 min at 40° C., cooled to ambient temperature,filtered and dried in vacuo in a lyophilization vessel immersed in a 60°C. oil bath overnight to afford the title compound [411 mg, 96%(uncorrected for residual solvents)] as a white solid. Final productpurity by HPLC was 98.07 area %. Residual solvents observed by ¹H NMRincluded TFE (2.03% w/w) and MeOAc (0.19% w/w).

Example 5. XRPD Pattern of a Hydrochloride Salt of Compound I fromMethano: 2-Propanol (75%: 25% v/v)

TABLE A FWHM Left Rel. Int. Pos. [°2θ] Height [cts] [°2θ] d-spacing [Å][%] 3.7579 3373.12 0.0768 23.51305 70.94 4.2659 4169.45 0.0895 20.7140987.69 6.5526 917.62 0.0640 13.48938 19.30 7.2841 697.14 0.0895 12.1364114.66 8.0800 85.65 0.0900 10.93359 1.80 8.5065 84.96 0.1535 10.394891.79 9.7875 1672.39 0.0895 9.03704 35.17 10.6288 355.31 0.0895 8.323617.47 12.0543 189.97 0.1535 7.34226 4.00 12.7809 419.79 0.1279 6.926438.83 13.3068 656.50 0.1151 6.65387 13.81 14.1827 1182.25 0.0895 6.2448624.86 14.5619 1327.04 0.1279 6.08305 27.91 15.0219 487.99 0.1023 5.8978310.26 16.1288 613.50 0.2303 5.49546 12.90 16.9425 569.74 0.1535 5.2333111.98 18.0211 4755.00 0.1151 4.92245 100.00 18.7982 2930.95 0.14074.72068 61.64 19.1243 583.81 0.1023 4.64092 12.28 19.6849 749.80 0.15354.51001 15.77 20.1376 596.39 0.1535 4.40963 12.54 20.5047 887.59 0.15354.33150 18.67 20.9553 2520.28 0.1151 4.23938 53.00 22.0163 1124.720.0895 4.03740 23.65 22.6867 1684.20 0.1279 3.91959 35.42 23.2292 904.360.1407 3.82926 19.02 24.0145 846.12 0.2047 3.70580 17.79 24.5746 412.170.1791 3.62258 8.67 25.1662 582.42 0.1279 3.53876 12.25 25.9049 650.360.1279 3.43950 13.68 26.4986 307.14 0.2558 3.36377 6.46 27.4092 275.340.1535 3.25405 5.79 27.9577 182.83 0.2047 3.19144 3.85 29.2173 173.310.3070 3.05666 3.64 30.4972 145.70 0.1535 2.93123 3.06 30.9418 102.650.0900 2.88773 2.16 31.7727 126.66 0.3070 2.81642 2.66 32.2938 161.360.1535 2.77215 3.39 33.4213 63.17 0.3070 2.68116 1.33

Example 6. XRPD Pattern of a Hydrochloride Salt of Compound I fromMethanol

TABLE B FWHM Left Rel. Int. Pos. [°2θ] Height [cts] [°2θ] d-spacing [Å][%] 3.7036 1966.63 0.0640 23.85759 72.77 4.4432 2477.30 0.1023 19.8874991.66 6.5634 704.32 0.0895 13.46726 26.06 7.4018 429.84 0.0512 11.9436615.90 9.7210 1158.88 0.1023 9.09875 42.88 10.6210 224.51 0.1023 8.329648.31 11.0920 133.97 0.1535 7.97704 4.96 12.7331 103.83 0.0900 6.946603.84 13.1598 415.98 0.1023 6.72787 15.39 14.0930 454.88 0.1023 6.2843916.83 14.7901 1040.30 0.0768 5.98973 38.49 16.7103 439.59 0.1535 5.3055216.27 17.1509 141.83 0.0900 5.16593 5.25 18.0233 2702.62 0.1023 4.92186100.00 18.5346 735.14 0.1023 4.78723 27.20 18.7979 1147.74 0.06404.72075 42.47 19.1239 789.29 0.0895 4.64101 29.20 19.5235 306.83 0.09004.54315 11.35 19.8085 422.15 0.1023 4.48213 15.62 20.1544 324.59 0.15354.40598 12.01 20.6070 478.45 0.1023 4.31022 17.70 20.8573 1011.67 0.06404.25907 37.43 21.3109 537.54 0.1535 4.16942 19.89 22.0189 619.33 0.23034.03694 22.92 22.6758 1060.70 0.1023 3.92146 39.25 23.0757 398.04 0.15353.85439 14.73 23.6744 38.83 0.0900 3.75515 1.44 23.9728 340.14 0.17913.71215 12.59 24.4777 293.52 0.1535 3.63671 10.86 25.7316 132.83 0.09003.45941 4.91 26.4407 215.92 0.2047 3.37100 7.99 27.7972 173.31 0.17913.20950 6.41 30.1162 99.20 0.2047 2.96744 3.67 32.1646 67.76 0.30702.78299 2.51

Example 7. XRPD Pattern of a Tosylate Salt of Compound I from Acetone

TABLE C Pos. FWHM Rel. Int. [°2Th.] Height [cts] [°2Th.] d-spacing [Å][%] 5.1621 2149.15 0.1663 17.11956 100.00 8.9359 438.04 0.2303 9.8963520.38 10.7664 184.28 0.2047 8.21754 8.57 13.3445 266.11 0.2047 6.6351712.38 14.3884 539.35 0.4605 6.15602 25.10 15.9863 161.64 0.2558 5.544127.52 17.2952 407.57 0.2558 5.12739 18.96 18.8443 420.10 0.2047 4.7092519.55 19.5521 362.22 0.1535 4.54033 16.85 21.0051 693.10 0.1535 4.2294332.25 23.2998 206.42 0.3582 3.81783 9.60 24.6506 86.12 0.5117 3.611594.01

Example 8. XRPD Pattern of a Mesylate Salt of Compound I from Acetone

TABLE D Pos. FWHM Rel. Int. [°2Th.] Height [cts] [°2Th.] d-spacing [Å][%] 5.4291 3079.02 0.0384 16.27808 100.00 8.0335 120.51 0.1535 11.005803.91 9.0773 115.67 0.2558 9.74250 3.76 10.8149 293.11 0.1279 8.180749.52 13.4288 415.08 0.1279 6.59369 13.48 14.7938 421.32 0.2558 5.9882213.68 15.7960 318.03 0.1791 5.61049 10.33 17.5693 431.98 0.1535 5.0480014.03 18.9795 376.14 0.1279 4.67599 12.22 19.6937 289.54 0.2047 4.507999.40 21.3056 720.36 0.2303 4.17044 23.40 22.2563 263.14 0.1535 3.994428.55 24.1299 237.45 0.7164 3.68833 7.71 25.7352 135.39 0.3070 3.461804.40 27.6495 84.81 0.4093 3.22631 2.75 30.9496 17.50 0.6140 2.88941 0.57

Example 9. XRPD Pattern of an Oxalate Salt of Compound I from Acetone

TABLE E FWHM Left Rel. Int. Pos. [°2θ] Height [cts] [°2θ] d-spacing [Å][%] 4.0824 291.09 0.1023 21.64476 10.42 7.2188 323.51 0.1279 12.2460011.58 7.7660 749.77 0.1023 11.38428 26.85 8.1685 455.55 0.0768 10.8242716.31 8.7856 126.66 0.1023 10.06525 4.54 10.1057 979.70 0.0768 8.7532735.08 11.7275 233.13 0.1023 7.54614 8.35 12.0519 307.19 0.1023 7.3437411.00 12.2864 248.60 0.1023 7.20411 8.90 12.7774 846.47 0.1023 6.9283730.31 13.2740 468.08 0.0768 6.67023 16.76 13.6796 175.54 0.1535 6.473366.29 14.4861 387.62 0.1151 6.11472 13.88 14.9214 326.40 0.1151 5.9373211.69 15.1377 150.25 0.0900 5.84810 5.38 15.5347 106.18 0.0900 5.699543.80 16.1829 161.09 0.1535 5.47721 5.77 17.7646 1786.25 0.1151 4.9929563.96 18.1548 399.21 0.1407 4.88652 14.30 18.5392 2792.57 0.0895 4.78604100.00 18.9751 574.37 0.1023 4.67707 20.57 19.9095 1049.25 0.10234.45962 37.57 20.3639 672.05 0.1535 4.36113 24.07 21.0328 208.25 0.20474.22393 7.46 21.9414 400.27 0.0900 4.04767 14.33 21.9676 539.10 0.10234.04624 19.30 22.2732 718.09 0.1535 3.99141 25.71 22.8116 257.00 0.15353.89841 9.20 23.3197 -104.04 0.0900 3.81146 −3.73 23.5325 291.26 0.12793.78060 10.43 24.0190 147.43 0.1535 3.70511 5.28 24.8019 132.93 0.30703.58990 4.76 25.2913 145.65 0.2303 3.52153 5.22 25.6339 162.92 0.20473.47524 5.83 26.6566 202.58 0.2047 3.34419 7.25 27.7426 83.70 0.15353.21570 3.00 28.2705 228.59 0.1279 3.15684 8.19 28.9314 78.20 0.09003.08365 2.80 29.2563 124.89 0.2047 3.05267 4.47 30.0846 91.09 0.15352.97049 3.26 30.7947 123.92 0.2047 2.90359 4.44 31.5899 127.84 0.15352.83229 4.58 31.9371 93.79 0.0900 2.79997 3.36 32.8553 71.65 0.20472.72605 2.57

Example 10. XRPD Pattern of a Benzoate Salt of Compound I f fromMethanol

TABLE F FWHM Left Rel. Int. Pos. [°2θ] Height [cts] [°2θ] d-spacing [Å][%] 3.6742 2073.90 0.0768 24.04811 100.00 4.4204 1347.38 0.0895 19.9901764.97 6.7177 829.29 0.0768 13.15834 39.99 7.0320 229.18 0.0768 12.5709211.05 8.2133 416.05 0.0895 10.76529 20.06 9.8583 662.53 0.0895 8.9723531.95 10.2604 109.77 0.0900 8.61446 5.29 12.3942 500.02 0.1023 7.1417024.11 13.2923 979.35 0.0895 6.66111 47.22 13.6972 1012.99 0.1023 6.4651048.84 14.1013 1154.57 0.1023 6.28071 55.67 14.6960 340.34 0.2047 6.0278816.41 15.7923 726.93 0.1023 5.61180 35.05 16.4926 394.77 0.1023 5.3750519.04 17.0775 509.25 0.0768 5.19226 24.56 17.2537 552.89 0.1279 5.1396126.66 18.0813 1729.49 0.1279 4.90621 83.39 18.4075 885.37 0.1279 4.8199942.69 18.9537 1288.35 0.1279 4.68231 62.12 19.5451 747.42 0.1279 4.5419436.04 19.7885 486.20 0.1023 4.48661 23.44 20.2166 505.95 0.1279 4.3925624.40 20.6718 1051.62 0.1407 4.29685 50.71 20.8819 961.73 0.1279 4.2541146.37 21.6381 545.53 0.1023 4.10712 26.30 22.2851 1623.89 0.1535 3.9893078.30 22.6611 298.77 0.0900 3.92072 14.41 23.0749 506.06 0.1535 3.8545324.40 24.3020 1218.36 0.1407 3.66260 58.75 24.7773 408.23 0.2047 3.5934119.68 25.5656 337.24 0.2558 3.48437 16.26 26.6658 206.54 0.1535 3.343059.96 27.2542 199.90 0.2558 3.27220 9.64 27.7067 201.69 0.1535 3.219789.72 28.3209 285.54 0.2558 3.15134 13.77 29.3217 120.66 0.2047 3.046025.82 30.1348 153.42 0.4093 2.96566 7.40 30.4172 195.25 0.1535 2.938769.41 31.1772 176.27 0.3070 2.86884 8.50 32.4960 59.94 0.7164 2.755362.89 33.6018 144.00 0.1791 2.66717 6.94

INCORPORATION BY REFERENCE

All of the U.S. patents and U.S. and PCT published patent applicationscited herein are hereby incorporated by reference.

EQUIVALENTS

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of theinvention are not necessarily encompassed by each embodiment of theinvention.

1. A crystalline form of a salt of Compound I,

(Boc-D-Arg-DMT-Lys(Boc)-Phe-NH₂; (I)), wherein said crystalline form hascharacteristic peaks in its XRPD pattern at values of two theta asdescribed in any one of Tables A′-F′; wherein Table A′ is: Pos. [°2θ] 03.7579 4.2659 6.5526 7.2841 8.0800 8.5065 9.7875 10.6288 12.0543 12.780913.3068 14.1827 14.5619 15.0219 16.1288 16.9425 18.0211 18.7982 19.124319.6849 20.1376 20.5047 20.9553 22.0163 22.6867 23.2292 24.0145 24.574625.1662 25.9049 26.4986 27.4092 27.9577 29.2173 30.4972 30.9418 31.772732.2938 33.4213

Table B′ is: Pos. [°2θ] 6 3.7036 4.4432 6.5634 7.4018 9.7210 10.621011.0920 12.7331 13.1598 14.0930 14.7901 16.7103 17.1509 18.0233 18.534618.7979 19.1239 19.5235 19.8085 20.1544 20.6070 20.8573 21.3109 22.018922.6758 23.0757 23.6744 23.9728 24.4777 25.7316 26.4407 27.7972 30.116232.1646

Table C′ is: Pos. [°2θ] 5.1621 8.9359 10.7664 13.3445 14.3884 15.986317.2952 18.8443 19.5521 21.0051 23.2998 24.6506

Table D′ is: Pos. [°2Th.] 5.4291 8.0335 9.0773 10.8149 13.4288 14.793815.7960 17.5693 18.9795 19.6937 21.3056 22.2563 24.1299 25.7352 27.649530.9496

Table E′ is: Pos. [°2θ] 4.0824 7.2188 7.7660 8.1685 8.7856 10.105711.7275 12.0519 12.2864 12.7774 13.2740 13.6796 14.4861 14.9214 15.137715.5347 16.1829 17.7646 18.1548 18.5392 18.9751 19.9095 20.3639 21.032821.9414 21.9676 22.2732 22.8116 23.3197 23.5325 24.0190 24.8019 25.291325.6339 26.6566 27.7426 28.2705 28.9314 29.2563 30.0846 30.7947 31.589931.9371 32.8553

and Table F′ is: Pos. [°2θ] 3.6742 4.4204 6.7177 7.0320 8.2133 9.858310.2604 12.3942 13.2923 13.6972 14.1013 14.6960 15.7923 16.4926 17.077517.2537 18.0813 18.4075 18.9537 19.5451 19.7885 20.2166 20.6718 20.881921.6381 22.2851 22.6611 23.0749 24.3020 24.7773 25.5656 26.6658 27.254227.7067 28.3209 29.3217 30.1348 30.4172 31.1772 32.4960 33.6018


2. A crystalline form of a salt of Compound I,

wherein said crystalline form has characteristic peaks in its XRPDpattern as described in any of the following spectra:


3. (canceled)
 4. A crystalline form of a hydrochloride salt of CompoundI,

wherein said crystalline form has characteristic peaks in its XRPDpattern at values of two theta (° 2θ) of: 3.8, 4.3, 9.8, 14.6, 18.0,18.8, 20.9, and 22.7.
 5. The crystalline form of claim 4, wherein saidcrystalline form has characteristic peaks in its XRPD pattern at valuesof two theta (° 2θ) of: 3.8, 4.3, 6.5, 7.3, 9.8, 13.3, 14.2, 14.6, 16.1,16.9, 18.0, 18.8, 19.1, 19.7, 20.1, 20.5, 20.9, 22.0, 22.7, 23.2, 24.0,25.2, and 25.9.
 6. A crystalline form of a hydrochloride salt ofCompound I,

wherein said crystalline form has characteristic peaks in its XRPDpattern at values of two theta (° 2θ) of: 3.7, 4.4, 6.6, 9.7, 14.8,18.0, 18.5, 18.8, 19.1, 20.9, and 22.7.
 7. The crystalline form of claim6, wherein said crystalline form has characteristic peaks in its XRPDpattern at values of two theta (° 2θ) of: 3.7, 4.4, 6.6, 7.4, 9.7, 10.6,13.2, 14.1, 14.8, 16.7, 18.0, 18.5, 18.8, 19.1, 19.5, 19.8, 20.1, 20.6,20.9, 21.3, 22.0, 22.7, 23.1, and 24.0.
 8. A crystalline form of atosylate salt of Compound I,

wherein said crystalline form has characteristic peaks in its XRPDpattern at values of two theta (° 2θ) of: 5.2, 8.9, 14.4, 17.3, 18.8,19.5, and 21.0.
 9. The crystalline form of claim 8, wherein saidcrystalline form has characteristic peaks in its XRPD pattern at valuesof two theta (° 2θ) of: 5.2, 8.9, 10.8, 13.4, 14.4, 16.0, 17.3, 18.8,19.5, 21.0, 23.3, and 24.6.
 10. A crystalline form of a mesylate salt ofCompound I,

wherein said crystalline form has characteristic peaks in its XRPDpattern at values of two theta (° 2θ) of: 5.4, 13.4, 14.8, 15.8, 17.6,19.0, and 21.3.
 11. The crystalline form of claim 10, wherein saidcrystalline form has characteristic peaks in its XRPD pattern at valuesof two theta (° 2θ) of: 5.4, 10.8, 13.4, 14.8, 15.8, 17.6, 19.0, 19.7,21.3, 22.3. 24.1, and 25.7.
 12. A crystalline form of an oxalate salt ofCompound I,

wherein said crystalline form has characteristic peaks in its XRPDpattern at values of two theta (° 2θ) of: 7.8, 10.1, 12.8, 17.8, 18.5,19.9, and 22.3.
 13. The crystalline form of an oxalate salt of claim 12,wherein said crystalline form has characteristic peaks in its XRPDpattern at values of two theta (° 2θ) of: 4.1, 7.2, 7.8, 8.1, 10.1,12.0, 12.8, 13.3, 14.5, 14.9, 17.8, 18.1. 18.5, 19.9, 20.4, 21.9, 22.0,22.3, and 23.5.
 14. A crystalline form of a benzoate salt of Compound I,

wherein said crystalline form has characteristic peaks in its XRPDpattern at values of two theta (° 2θ) of: 3.7, 4.4, 14.1, 18.1, 18.9,20.7, 22.3, and 24.3.
 15. The crystalline form of claim 14, wherein saidcrystalline form has characteristic peaks in its XRPD pattern at valuesof two theta (° 2θ) of: 3.7, 4.4, 6.7, 9.9, 13.3, 13.7, 14.1, 15.8,17.2, 18.1, 18.4, 18.9, 19.5, 20.7, 20.9, 21.6, 22.3, and 24.3.
 16. Amethod of making Compound II, the method comprising deprotectingcompound (I),

or a salt thereof, thereby making compound (II)

or a salt thereof.
 17. The method of claim 16, wherein the step ofdeprotecting comprises: a. preparing a mixture of a crystalline form ofcompound (I), triisopropylsilane, and 2,2,2-trifluoroethanol; and b.adding concentrated hydrochloric acid to the mixture.
 18. The method ofclaim 17, wherein the mixture is a slurry.